TITLE: Tissue NET-seq  (native elongating transcript sequencing)
AUTHORS: Mihaela   Bozukova, Peter Tessarz

[DESCRIPTION]
Quantifying crucial steps in gene regulation during transcription elongation, such as promoter-proximal pausing, requires high resolution methods to map the transcription machinery across the genome. Native Elongating Transcript sequencing (NET-seq) interrogates the 3' ends of nascent RNA through sequencing, providing a direct visualization of RNA Polymerase II (Pol II) positions genome-wide with strand specificity and single nucleotide resolution. As NET-seq does not require previous labeling of mRNA, it is ideally-suited for measuring nascent transcription in tissues. In brief, actively transcribing Pol II together with the nascent RNA is quantitatively purified from cells by cellular fractionation. To prevent run-on transcription, fractionation is performed in the presence of the potent Pol II inhibitor α-amanitin, which prevents NTP recognition and catalysis by the Pol II trigger loop. Then, the isolated nascent RNA is fragmented, converted into cDNA and processed into a sequencing library employing a minimal number of PCR cycles. The library preparation method is designed in a way that upon random fragmentation of the nascent transcripts, only the 3’ ends of nascent transcripts carrying a free hydroxy-group are sequenced. This reveals the position of transcriptionally engaged Pol II at nucleotide resolution.
We modified the original NET-seq protocol (Mayer & Churchman, 2016) for application in murine liver tissue and named this new adaptation of the protocol tissue NET-seq (tNET-seq). The main change in the protocol pertains to the nuclei isolation. Fresh liver tissue were placed in ice-cold PBS, cut into small pieces and homogenized in nuclei isolation buffer (Sigma, NUC101) using a Dounce tissue homogenizer. 
A published analysis pipeline for NET-seq can be found at the Churchman lab Github.


[BEFORE_START]
The following steps are based on the protocol described in (Mayer and Churchman 2016), but modified to work in tissues. The main change in the protocol pertains to the nuclei isolation.

[GUIDELINES]
Notes
1. Irritant, handle with care and dispose of the waste according to institutional regulations.
2. Toxic, handle with care and dispose of the waste according to institutional regulations.
3. Irritant, harmful, and hazardous to the environment: handle with care and dispose of the waste according to institutional regulations.
4. Toxic, corrosive, and an irritant: handle with care and dispose of waste according to institutional regulations.
5. When the fractionation protocol is being tested on a new cell type it is recommended that the cellular fractions are checked for subcellular marker proteins in order to confirm that the fractionation is working. To do this, probe the WB membrane with antibodies raised against GAPDH (cytoplasm), the U1 snRNP70 (nucleoplasm) and histone 2B (chromatin).
6. The CTD specificities of these Pol II–specific antibodies have been extensively characterized, so we recommend using these specific antibodies when testing the fractionation protocol.  
7. Benzonase (250 U) can be used to resuspend the sticky chromatin pellet in solution (for the WB control, Box 1).
8. Flammable, corrosive, and an irritant: handle with care and dispose of waste according to institutional regulations
9. Volatile, toxic, and an irritant: handle with care and dispose of waste according to institutional regulations.
10. It is important to use the miRNeasy mini kit (or miRNeasy micro kit for small cell numbers) instead of the RNeasy mini kit to avoid RNA length biases. Use personal protective equipment when handling reagents in the kits; the RWT buffer is corrosive and an irritant, and the QIAzol lysis reagent contains phenol and is toxic and corrosive. Dispose of waste according to institutional regulations.
11. Handle sharps with care and dispose according to institutional regulations.
12. Keep track of the enzyme lot; ligase activity can vary between different batches.
13. Using an N10 linker increases the dynamic range of the NET-seq library and decreases PCR duplicates (compared to the N6 linker used in (Mayer and Churchman 2016)).
14. Use personal protective equipment when handling reagents in the kit; buffers are irritants. Dispose of waste according to institutional regulations.
15. Nucleic acid stains are usually mutagenic; use personal protective equipment when handling.  SYBR Gold stain is flammable; dispose of waste according to institutional regulations.
16. Corrosive and an irritant: handle solutions with care and dispose of waste according to institutional regulations. Limit the freeze-thaw cycles for this reagent.
17. Toxic and hazardous to the environment: handle with care and dispose of waste according to institutional regulations.
18. Highly flammable, volatile, and an irritant: handle solutions with care and dispose of waste according to institutional regulations.
19. NET-seq has been successfully carried out on several human cell lines (e.g. K562, HeLa S3, HEK293T, and MOLT-4 cells), including challenging cell types such as neurons. The NET-seq protocol has also been successfully applied to other cell types such as Drosophila cells (S2 and S2R+ cells) and mouse cells (NIH 3T3 cells) and others.
20. Perform a western blot control  in parallel with the sample, or prior to performing fractionation on the sample to ensure the cellular fractionation protocol is optimized for the specific tissue being used.  The western blot control is a separate sample from the sample being prepared to make a sequencing library.
21. We used roughly 1/2 of a liver per sample.
22. If there is a small amount of supernatant remaining in the tube after the greater quantity has been removed, it is helpful to use a small pipette tip to get rid of the residual supernatant.
23. For more effective layering, tilt the tube at a 45 degree angle and pipette lysate onto the side of the tube.
24. Some chromatin pellets can be difficult to resuspend. If you find this to be the case, you can either increase the volume of the CRS (up to 300 µl) and try to solubilize the chromatin pellet again or you can proceed directly to the RNA extraction (Steps section 1.3.3) by adding Qiazol to your chromatin pellet (which is in 50 µl of CRS).
25. Alternatively, the chromatin pellet can also be solubilized by gentle vortexing. 
26. The expected absorbance at (A260/A280) ratio is ~2.0, indicative of highly purified RNA.
27. The fragmentation time needs to be adjusted whenever a new batch of alkaline fragmentation solution is applied (or when the solution has not been used for long periods of time -i.e. months). An over fragmentation or under fragmentation of the nascent RNA pool can lead to systematic biases. In a typical experiment RNA is fragmented between 10 and 40 min at 95 °C.
28. The un-ligated oGAB11 control is only used for visualizing the efficiency of the ligation reaction by comparing un-ligated oGAB11 to ligated oGAB11. After visualization of the gel, un-ligated oGAB11 control is not subjected to any further steps in the protocol.
29. Use a needle and syringe to flush out the excess urea that may settle in the gel wells prior to loading samples.
30. The efficiency of the linker ligation is usually >95%, the efficiency of the ligation is monitored using the oGAB11 control by PAGE (see Figure 2).
31. If no cDNA smear is observed continue to the PCR step; sometimes PCR product will be visible even when the RT product is not visible by eye. Other possible reasons for not observing the RT product are that the linker ligation was inefficient or the reverse transcriptase enzyme lost activity.  One way to prevent these problems is to replace the ligation reaction and RT reaction reagents every 4 months.
32. Empty circles are circularized DNA molecules that arise from un-extended RT primers, and hence they do not contain any information about the original nascent RNA.
33. Do not dilute the NET-seq library for the quantification and characterization step if less than 1 µg of starting material was used to construct the sequencing library.



References
Adelman K, Lis JT. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.Nat Rev Genet. 2012;13(10):720-731. doi:10.1038/nrg3293

Boswell SA, Snavely A, Landry HM, Churchman LS, Gray JM, Springer M. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.Nat Chem Biol. 2017;13(5):501-507. doi:10.1038/nchembio.2317

Cai H, Luse DS. Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes.J Biol Chem. 1987;262(1):298-304.

Churchman LS, Weissman JS. Native elongating transcript sequencing (NET-seq).Curr Protoc Mol Biol. 2012;Chapter 4:. doi:10.1002/0471142727.mb0414s98

Ietswaart R, Wu Z, Dean C. Flowering time control: another window to the connection between antisense RNA and chromatin. Trends Genet. 2012 Sep;28(9):445-53. doi: 10.1016/j.tig.2012.06.002. Epub 2012 Jul 10. PMID: 22785023.

Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012 Jul 26;7(8):1534-50. doi: 10.1038/nprot.2012.086. PMID: 22836135; PMCID: PMC3535016.

Jin Y, Eser U, Struhl K, Churchman LS. The Ground State and Evolution of Promoter Region Directionality. Cell. 2017 Aug 24;170(5):889-898.e10. doi: 10.1016/j.cell.2017.07.006. Epub 2017 Aug 10. PMID: 28803729; PMCID: PMC5576552.

Margaritis T, Holstege FC. Poised RNA polymerase II gives pause for thought. Cell. 2008 May 16;133(4):581-4. doi: 10.1016/j.cell.2008.04.027. PMID: 18485867.

Mayer A, Churchman LS. Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing.Nat Protoc. 2016;11(4):813-833. doi:10.1038/nprot.2016.047

Mayer A, di Iulio J, Maleri S, et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution.Cell. 2015;161(3):541-554. doi:10.1016/j.cell.2015.03.010

Rinn JL, Chang HY. Genome regulation by long noncoding RNAs.Annu Rev Biochem. 2012;81:145-166. doi:10.1146/annurev-biochem-051410-092902

Winter GE, Mayer A, Buckley DL, et al. BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.Mol Cell. 2017;67(1):5-18.e19. doi:10.1016/j.molcel.2017.06.004

Wuarin J, Schibler U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.Mol Cell Biol. 1994;14(11):7219-7225. doi:10.1128/mcb.14.11.7219-7225.1994

[STEPS]
SECTION: Tissue homogenization and cell fractionation
1. Place the fresh liver tissue in ice-cold PBS, cut into smaller pieces and homogenize in 3 ml of nuclei isolation buffer using a Dounce tissue homogenizer (Wheaton). After complete homogenization, transfer the sample to a Falcon tube. Add 3 ml of nuclei isolation buffer to the remaining pieces in the tissue homogenizer, homogenize further and transfer the remnants also to the tube. Following an incubation on ice for 5 min, pass the samples through a 70-μm cell strainer and collect the nuclei by centrifuging at 500 x g for 20 min.
Resuspend the nuclei pellet in 4 ml of nuclei isolation buffer. After incubation on ice for 5 min, collect the nuclei by centrifuging at 500 x g for 5 min. To remove cytoplasmic remnants, wash the nuclei pellet with 1600 μl nuclei wash buffer and centrifuge at 1,150 x g for 5 min. Repeat the washing with 800 μl nuclei wash buffer. Then, gently resuspend the pellet in 200 µl of glycerol buffer using a cut 1,000-µl tip and transfer the suspension to a new 1.5-ml RNase-free microcentrifuge tube. To lyse nuclei, add 400 µl of nuclei lysis buffer, mix samples by pulsed vortexing and incubate on ice for 20 min. After assessing nuclei lysis of a small aliquot under a light microscope, centrifuge the samples at 18,500 x g for 2 min. Completely remove the supernatant (nucleoplasmic fraction) and resuspend the chromatin pellet in 50 µl chromatin resuspension solution.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Add 20 µl of 2× TBU denaturing sample buffer to the cDNA sample and the oGAB11 cDNA control, mix well.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Denature the cDNA sample, oGAB11 cDNA control and DNA control ladder for 3 min at 95 °C in a Thermomixer.  Place the samples on ice.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Prerun a 10% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35).
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Load gel (20 uL per lane, each sample will be split into two lanes) with the cDNA sample, the oGAB11 cDNA control and the DNA ladder. Separate by PAGE at 200 V for 65 min.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Visualize the gel under blue/UV light.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. For the cDNA sample, excise region between ~85 and 160 nt (use oGAB11 as baseline and cut up ~70 nt). For the cDNA oGAB11 control, excise a narrow band ~85 nt. If no cDNA smear is observed see Note 37.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. Perform rapid gel extraction on the excised gel (see Box 3). There will be two gels for one sample since each sample was split into two lanes, combine the two gels from one sample into a single tube.
SECTION: 3.4.6 Size select cDNA and gel extraction (Figure 3)
None. After rapid gel extraction is performed, use the Oligo kit (Zymo) and elute cDNA from column with 15 uL of 10 mM Tris-HCl (pH 8.0) (at this point, the cDNA can be stored indefinitely at −20 °C). Alternatively, cDNA can be prepared by precipitation (see Note 33).
SECTION: 3.4.7 cDNA Circularization (Figure 4)
41. Prepare circularization mix and store it on ice.
SECTION: 3.4.7 cDNA Circularization (Figure 4)
42. In a 0.2-ml RNase-free PCR tube, add 4 µl of circularization mix to 15 µl of the cDNA sample and the oGAB11 cDNA control sample, mix well.
SECTION: 3.4.7 cDNA Circularization (Figure 4)
43. Add 1 µl of CircLigase (100 U/µl) and mix.
SECTION: 3.4.7 cDNA Circularization (Figure 4)
44. Incubate the CircLigase reaction for 60 min at 60 °C and then 10 min at 80 °C in a thermal cycler. Circularized cDNA can be stored indefinitely at −20 °C.
SECTION: 3.4.7 cDNA Circularization (Figure 4)
45. Optional: at this point a specific depletion of highly abundant mature RNAs can be performed (see Note
SECTION: 3.4.8 PCR amplification (Figure 4)
46. Perform PCR amplification test (Box 4)
SECTION: 3.4.8 PCR amplification (Figure 4)
47. Prepare PCR mix for four PCR amplification reactions for both the cDNA sample and the oGAB11 control cDNA sample. Mix well and store on ice.
SECTION: 3.4.8 PCR amplification (Figure 4)
48. For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
SECTION: 3.4.8 PCR amplification (Figure 4)
49. Add 1 µl of circularized cDNA and mix it well.
SECTION: 3.4.8 PCR amplification (Figure 4)
50. Perform PCR amplifications (as shown in Table 2) with determined optimal number of PCR cycles (see Box 4).
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
51. Prepare DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of DNase-free H2O. Add 2 µl of 6× DNA loading dye and mix well.
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
52. Add 3.4 µl of 6× DNA loading dye to each PCR sample tube and mix well.
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
53. Load gel with the PCR products and DNA control ladder. Separate by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
54. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
55. Visualize the gel under blue/UV light.
SECTION: 3.4.9 PCR size selection and gel extraction (Figure 4)
56. For each sample, excise the band from the gel that contains the PCR product. The NET-seq library is a broad band that runs at ~150 nt (use oGAB as baseline and cut up ~50 nt). Avoid contamination from the lower band that runs at ~120 nt, representing PCR product from empty circles (see Note 39). The oGAB11 control sample is not subjected to this step, or any further steps.
SECTION: 3.4.10 PCR product cleanup
57. Perform the first four steps of rapid gel extraction (see Box 3)
SECTION: 3.4.10 PCR product cleanup
58. After centrifugation, add 670 µl of DNA soaking buffer and mix.
SECTION: 3.4.10 PCR product cleanup
59. Incubate the samples overnight at room temperature in a Thermomixer (set at 1,500 r.p.m.).
SECTION: 3.4.10 PCR product cleanup
60. Use a cut 1,000 µl pipette tip to transfer the gel slurry into a microcentrifuge tube filter.
SECTION: 3.4.10 PCR product cleanup
61. Centrifuge at 20,000g for 3 min at room temperature. Transfer the eluate to a new 1.5 ml microcentrifuge tube.
SECTION: 3.4.10 PCR product cleanup
62. Precipitate the NET-seq library by adding 2 µl of 15 mg/ml GlycoBlue and 680 µl of isopropanol. Mix well then incubate the precipitations at −20 °C for ≥1 h (the DNA precipitations can be stored at −20 °C overnight).
SECTION: 3.4.10 PCR product cleanup
63. Centrifuge at 20,000g for 30 min at 4 °C to pellet the RNA.
SECTION: 3.4.10 PCR product cleanup
64. Remove the supernatant and wash the pellet with 750 µl of 80% (vol/vol) ice-cold ethanol.
SECTION: 3.4.10 PCR product cleanup
65. Spin the sample at 20,000g for 2 min at 4 °C, and then remove the supernatant. Air-dry the RNA pellet for 10 min at room temperature.
SECTION: 3.4.10 PCR product cleanup
66. Resuspend the NET-seq library in 7 - 10 µl of Tris-HCl (10 mM, pH 8.0). The DNA sequencing library can be stored indefinitely at −20 °C. 
SECTION: 3.4.11 Quantification and characterization of NET-seq library (Figure 4)
67. Prepare a 1:5 dilution of the NET-seq library by adding 1 µl of the NET-seq library to 4 µl of Tris-HCl (10 mM, pH 8.0); mix well (see Note 40).
SECTION: 3.4.11 Quantification and characterization of NET-seq library (Figure 4)
68. Use 1 µl of the diluted NET-seq library for quantification with the Qubit fluorometer using the Qubit dsDNA HS assay kit. Prepare the sample and perform the measurement according to the manufacturer’s protocol (see Note 41).
SECTION: 3.4.11 Quantification and characterization of NET-seq library (Figure 4)
69. Use 1 µl of the diluted NET-seq library for characterization on the Agilent Bioanalyzer; use the high-sensitivity DNA analysis kit according to the manufacturer’s instructions.
SECTION: 3.4.11 Quantification and characterization of NET-seq library (Figure 4)
70. Sequence using oLSC006. Sequence the human NET-seq library from the 3′ end (single-end sequencing) on the Illumina platform using oLSC006 (Table 1) as a custom sequencing primer. Perform sequencing according to the manufacturer’s instructions. NET-seq libraries are typically sequenced on MiSeq, HiSeq or NextSeq next-generation sequencing platforms. A reasonable coverage is obtained with 100–200 million reads.
SECTION: 3.5 Bioinformatics analysis (Figure 5)
71. Below we describe in brief the custom bioinformatics pipeline (see Figure 5) that is used to generate NET-seq coverage files. The scripts are available at http://github.com/churchmanlab/MiMB2019NETseq . See Note 42 for a description of the improvements relative to our previous pipeline (Mayer et al. 2015).
SECTION: 3.5.1 Alignment of sequencing reads
72. Assess read quality control with FastQC.
SECTION: 3.5.1 Alignment of sequencing reads
73. Displace 5’ end random barcode sequence from sequence to read identifier line and save as a separate fastq file. This barcode serves as a unique molecular identifier (UMI) and originates from the linker (see Table 1).
SECTION: 3.5.1 Alignment of sequencing reads
74. Align fastq files with and without UMI sequences separately to a human reference genome using STAR (See Note 43). In case a Drosophila spike-in was used, concatenate the human and Drosophila fasta files to generate a combined reference genome.
SECTION: 3.5.2 Filter out read alignments that do not reflect RNA polymerase position.
75. Remove alignments resulting from RT mispriming events, i.e. cases where RT priming did not occur at the complementary sequence site on the ligated linker but instead at a position within the RNA fragment. The resulting reads can be identified as they lack the random UMI sequence encoded on the linker and therefore align well to the reference genome both with and without UMI displacement: when the STAR alignment score of a read with the UMI sequence equals the UMI length (10nt) plus the alignment score from that read without UMI.
SECTION: 3.5.2 Filter out read alignments that do not reflect RNA polymerase position.
76. Remove alignments that map to multiple genomic locations by filtering for their MAPQ value (< 50) using samtools.
SECTION: 3.5.2 Filter out read alignments that do not reflect RNA polymerase position.
77. Remove alignments resulting from PCR duplication events, i.e. cases of multiple amplicons that originate from the same RNA fragment. The resulting multiple reads are bioinformatically indistinguishable as their alignments share the same position, strand, chromosome, UMI sequence and CIGAR score. To filter out these PCR duplicates, we maintain only one of the alignments.
SECTION: 3.5.2 Filter out read alignments that do not reflect RNA polymerase position.
78. Remove alignments that could arise from splicing intermediates, i.e. 3’ ends of annotated exons and introns.
SECTION: 3.5.3 Determine RNA polymerase coverage genome-wide from read alignments.
79. Use HTseq to generate a coverage (bedgraph) file containing the read counts at a base-pair resolution over the reference genome as determined from the 5’ end of the read alignments (without UMI) on the opposite strand. The resulting positions correspond to the 3’ ends of nascent RNA and thereby map RNA polymerase positions. The coverage files will be used for a variety of downstream bioinformatics analyses such as visual inspection (e.g. using IGV), metagene profiling (e.g. Bedtools), differential expression (e.g. DEseq2).
SECTION: Box 1 | Western blot control: checking cell fractionation
80. The western blot control is used to  determine whether or not the cellular fractionation protocol has been executed  successfully. The efficiency of the fractionation can be quantified by  measuring the amount of elongating Pol II captured in the chromatin fraction  (ideally≥95%). When the fractionation  protocol is being performed for the first time or being tested on a new cell  type, the cytoplasm, nucleoplasm, and chromatin fractions should be checked  for subcellular marker proteins (see Note 5) to confirm the fractionation is  working (see Note 20).
SECTION: Box 1 | Western blot control: checking cell fractionation
81. Adjust the nucleoplasmic (from 3.3.2) and chromatin fraction (from 3.3.2) volumes to the cytoplasmic fraction (from 3.3.1) volume by adding 1× PBS. Mix each fraction well and ensure the chromatin pellet is completely resuspended (see Note 7).
SECTION: Box 1 | Western blot control: checking cell fractionation
82. Add 50 µl of 2× SDS buffer (see section 2.3.2 step 7) to 50 µl of each subcellular fraction and boil the samples at 95 °C for 5 min (at this point, samples can be stored for months at −20 °C).
SECTION: Box 1 | Western blot control: checking cell fractionation
83. Separate samples by standard SDS-PAGE (loading 10 µl of each boiled sample per lane is usually sufficient).
SECTION: Box 1 | Western blot control: checking cell fractionation
84. Probe the membrane overnight at 4 °C with Pol II CTD Ser2-P antibody (3E10, 1:1,000 dilution) and Pol II CTD Ser5-P antibody (3E8, 1:1,000 dilution). These antibodies target transcribing RNA Pol II (see Note 6).
SECTION: Box 2 | Fragmentation test
85. Performing RNA fragmentation results in a homogeneous RNA pool which helps to avoid length biases in any downstream enzymatic reactions. The fragmentation test can be performed using total RNA (or any easily-obtained RNA sample). Once the appropriate fragmentation time is determined (where most of the RNA molecules range from 40 –110 nt), fragmentation can be performed on the nascent RNA (obtained from cellular fractionation) using the determined time (Figure 2); the size range of the nascent RNA and total RNA pools do not vary enough for the fragmentation time to be different. 
SECTION: Box 2 | Fragmentation test
86. Take 9 samples of RNA from a total RNA sample (or any other easily obtained RNA sample), each sample should be 20 µl in volume and contain ~1ug of RNA. Add 20 µl of 2× alkaline fragmentation solution to each sample and mix well (see Note 32); fragmentation is not performed for the oGAB11 control.
SECTION: Box 2 | Fragmentation test
87. Fragment each RNA sample at 95 °C in a thermal cycler for the following times: 0, 15, 20, 25, 30, 35, 40, 45, and 50 minutes.
SECTION: Box 2 | Fragmentation test
88. To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).
SECTION: Box 2 | Fragmentation test
89. Prepare RNA ladder (denoted as L in figure) by adding 1.0 µl of RNA control ladder to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and mix well.
SECTION: Box 2 | Fragmentation test
90. Prepare all RNA samples by adding 1.0 µl of 2× TBU denaturing sample buffer to each sample, mix well.
SECTION: Box 2 | Fragmentation test
91. Denature the RNA sample and RNA ladder for 2 min at 80 °C. Place the samples on ice.
SECTION: Box 2 | Fragmentation test
92. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35).
SECTION: Box 2 | Fragmentation test
93. Load gel with the RNA ladder and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
SECTION: Box 2 | Fragmentation test
94. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: Box 2 | Fragmentation test
95. Visualize the gel under blue/UV light. The optimal fragmentation time is when most RNA molecules are in the required size range, between 40 and 110 nt (the optimal fragmentation time in the figure below is between 25 and 30 minutes).
SECTION: Box 3 | Rapid gel extraction
96. This protocol for rapidly extracting RNA or cDNA from a polyacrylamide TBE-urea gel is similar to the gel extraction protocol described by the Weissman laboratory (Ingolia et al. 2012; Churchman and Weissman 2012).
SECTION: Box 3 | Rapid gel extraction
97. Use a 21G needle to pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube.
SECTION: Box 3 | Rapid gel extraction
98. Place the pierced 0.5-ml tube into a 1.5-ml RNase-free microcentrifuge tube.
SECTION: Box 3 | Rapid gel extraction
99. Put the gel slice (from fragmentation, RT, or PCR) into the pierced 0.5-ml tube.
SECTION: Box 3 | Rapid gel extraction
100. Centrifuge the nestled tubes, containing the gel slice, at 20,000g for 4 min at room temperature.
SECTION: Box 3 | Rapid gel extraction
101. Add 200 µl of RNase-free H2O to the chopped-up gel and mix.
SECTION: Box 3 | Rapid gel extraction
102. Incubate the sample mixture for 10 min at 70 °C in a Thermomixer.
SECTION: Box 3 | Rapid gel extraction
103. Vortex the mixture for 30 s at medium intensity.
SECTION: Box 3 | Rapid gel extraction
104. Cut the tip off of a 1,000-µl pipette tip and transfer the gel mixture into a microcentrifuge tube filter.
SECTION: Box 3 | Rapid gel extraction
105. Centrifuge the mixture at 20,000g for 3 min at room temperature.
SECTION: Box 4 | PCR amplification test
106. The NET-seq library is PCR amplified using an Illumina index forward primer (Barcode primer, see Table 1) and a reverse primer (oNTI231; Table 1) specific to NET-seq (Figure 4). For each NET-seq library preparation, the minimum number of PCR cycles needed to obtain a sequencing library must to be determined. The NET-seq library is then amplified with the determined minimal amount of PCR cycles; this avoids overamplification which can result in unwanted products such as PCR duplicates.
SECTION: Box 4 | PCR amplification test
107. Prepare the PCR master mix (see section 2.4.8 step 4) for four pilot PCR amplifications for each cDNA sample and the oGAB11 cDNA control sample (denoted as O in figure). Mix well and store it on ice.
SECTION: Box 4 | PCR amplification test
108. For each PCR sample, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
SECTION: Box 4 | PCR amplification test
109. Add 1 µl of circularized cDNA and mix it well.
SECTION: Box 4 | PCR amplification test
110. Perform PCR pilot amplifications (described in Table 2). Remove one PCR tube for each sample at the end of the extension step after 6, 8, 10 and 12 amplification cycles.
SECTION: Box 4 | PCR amplification test
111. Add 3.4 µl of 6× DNA loading dye to each tube and mix well.
SECTION: Box 4 | PCR amplification test
112. Prepare DNA control ladder (denoted as L in figure) by adding 1.0 µl of ladder to 9 µl of DNase-free H2O. Then add 2 µl of 6× DNA loading dye and mix well.
SECTION: Box 4 | PCR amplification test
113. Separate the PCR products and the DNA control ladder by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.
SECTION: Box 4 | PCR amplification test
114. Stain the gel in 50 ml of gel staining solution (see section 2.4.8) for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: Box 4 | PCR amplification test
115. Visualize the gel under blue/UV light and identify the optimal PCR amplification cycles for each cDNA sequencing library. The optimal PCR amplification cycles for the NET-seq library, which runs at ~150 nt, is determined by the presence of a clear band at ~150 nt and no higher-molecular-weight PCR products (the optimal number of cycles in the figure below is between 6 and 8).

SECTION: 1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.
None. Spray down all worksurfaces, pipettes, etc. with RNase away.
SECTION: 1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.
None. Prepare CLB, SB, NWB, GB, NLB, and CRS buffers (see Materials section 1.1 for buffer components) and store all solutions on ice.
SECTION: 1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.
None. Make sure tabletop centrifuge is at 4 °C.
SECTION: 1.2 Preparation of cells for fractionation
None. Cellular fractionation has been successfully performed on several different cell types (see Note 19 in the Guidelines section). The protocol was originally optimized for HeLa S3 or HEK293T cells (Mayer and Churchman 2016). When fractionating other cell types optimization may be required (see Note 20). Cells are ideally grown according to internationally recognized guidelines (such as those suggested by ENCODE) and handled according to the supplier’s instructions (see Note 21 in the Guidelines section).
SECTION: 3.2.1 Washing and counting cells
None. The following steps are for suspension cells, if working with adherent cells see Note 22 for steps on how to prepare the cells.
SECTION: 3.2.1 Washing and counting cells
None. Count out 1×107 cells as input for each fractionation, including a western blot control sample (see Note 23). If cell numbers are limited, fractionation can be performed on fewer cells (see Note 24).
SECTION: 3.2.1 Washing and counting cells
None. Collect cells by centrifugation at 500g for 2 min at room temperature.
SECTION: 3.2.1 Washing and counting cells
None. Wash with 10 ml of PBS (room temperature).
SECTION: 3.2.1 Washing and counting cells
None. Collect cells by centrifugation at 500g for 2 min at room temperature.
SECTION: 3.2.1 Washing and counting cells
None. Add spike-in cells (Drosophila S2, used for normalization) to the sample cells collected by centrifugation (the addition of a spike-in is optional, see Note 25).
SECTION: 3.2.1 Washing and counting cells
None. Repeat wash (step 3) and centrifugation (step 4).
SECTION: 3.2.1 Washing and counting cells
None. Remove supernatant by aspiration. It is important to remove the supernatant completely to avoid diluting the lysis buffer (see Note 26).
None. 
SECTION: 3.3.1 Isolation of nuclei fraction
1. Using a cut P1000 pipette tip, add 200 µl of CLB to cells and mix by pipetting the sample up and down ten times. Transfer sample to an RNase-free 1.5 ml microcentrifuge tube using a cut 1,000 µl pipette tip.
SECTION: 3.3.1 Isolation of nuclei fraction
2. Incubate the cell lysate on ice for 5 min.
SECTION: 3.3.1 Isolation of nuclei fraction
3. During incubation period, pipette 500 µl of SB to an RNase-free 1.5 ml microcentrifuge tube.
SECTION: 3.3.1 Isolation of nuclei fraction
4. After incubation period, use a cut 1,000 µl pipette tip to layer the cell lysate sample onto the SB (see Note 27).
SECTION: 3.3.1 Isolation of nuclei fraction
5. Centrifuge layered lysate at 16,000g for 10 min at 4 °C to collect nuclei.
SECTION: 3.3.1 Isolation of nuclei fraction
6. Pipette off supernatant completely from the nuclei pellet (supernatant contains the cytoplasmic fraction, retain for western blot, described in Box 1)
SECTION: 3.3.1 Isolation of nuclei fraction
7. Wash nuclei pellet with 800 µl of NWB (either by inverting the tube or pipetting up and down).
SECTION: 3.3.1 Isolation of nuclei fraction
8. Collect cell nuclei by centrifugation at 1,150g for 1 min at 4 °C.
SECTION: 3.3.1 Isolation of nuclei fraction
9. Pipette off supernatant from the nuclei pellet. It is important to remove the supernatant completely in order to remove cytoplasmic mature RNAs (see Note 26).
SECTION: 3.3.2 Isolation of chromatin fraction
10. Add 200 µl of GB to the nuclei. Use a cut 1,000 µl tip to resuspend the washed nuclei pellet by pipetting up and down.
SECTION: 3.3.2 Isolation of chromatin fraction
11. Transfer the nuclei suspension to a new 1.5 ml RNase-free microcentrifuge tube.
SECTION: 3.3.2 Isolation of chromatin fraction
12. Layer 200 µl of NLB onto nuclei suspension and mix by pulsed vortexing.
SECTION: 3.3.2 Isolation of chromatin fraction
13. Incubate the mixture on ice for 2 min.
SECTION: 3.3.2 Isolation of chromatin fraction
14. Centrifuge at 18,500g for 2 min at 4 °C to collect the chromatin pellet.
SECTION: 3.3.2 Isolation of chromatin fraction
15. Pipette off supernatant from the chromatin pellet. It is important to completely remove the supernatant in order to remove nucleoplasmic RNAs (retain supernatant for the nucleoplasm fraction of western blot, described in Box 1).
SECTION: 3.3.2 Isolation of chromatin fraction
16. Add 50 µl CRS to the chromatin pellet, transfer pellet in CRS to a new 1.5 ml tube and then resuspend pellet in CRS (see Note 28). Perform this step on the experimental sample (for the preparation of nascent RNA, section 3.3.3) and the control sample (for the western blot analysis, as described in Box 1).
SECTION: 3.3.3 Preparation of nascent RNA
17. Add 700 µl of QIAzol (lysis reagent in miRNeasy mini kit, Qiagen) to the resuspended chromatin (see Note 10 re QIAzol).
SECTION: 3.3.3 Preparation of nascent RNA
18. Solubilize chromatin pellet by slowly pipetting up and down using a 1-ml syringe with a 22G needle (see Note 29). Mix very carefully (to avoid spilling the sample) until the solution is homogeneous.
SECTION: 3.3.3 Preparation of nascent RNA
19. Prepare RNA using the miRNeasy mini kit (or miRNeasy micro kit for small cell numbers, see section 2.3.3 step 2) according to the manufacturer’s instructions, including optional DNase treatment.
SECTION: 3.3.3 Preparation of nascent RNA
20. Measure the prepared RNA quantity and quality using a NanoDrop (see Note 30). The RNA yield is usually in the range of 20–30 µg (this can vary quite a lot depending on the type of cells used). If fractionation was performed on less than 1×107 cells (or RNA yield is less than 20 µg) see Note 24 re RNA amount needed to construct sequencing library. Store the isolated RNA at −80 °C (can be stored for months) until ready to construct sequencing library.
SECTION: 3.4 Constructing DNA sequencing library from isolated nascent RNA (Figure 1b)
21. Spray down all work surfaces, pipettes, etc. with RNase away
SECTION: 3.4.1 Linker ligation (Figure 2)
22. Denature the RNA sample (from the cellular fractionation, section 3.3.3) and 5 µl of the oGAB11 control (10 µM; Table 1) for 2 min at 80 °C in a Thermomixer. Place on ice.
SECTION: 3.4.1 Linker ligation (Figure 2)
23. Prepare linker ligation mix for each RNA sample and for oGAB11 in 0.2-ml RNase-free PCR tubes. Mix until reaction is homogeneous, poor mixing will negatively affect the ligation efficiency.
SECTION: 3.4.1 Linker ligation (Figure 2)
24. Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.
SECTION: 3.4.1 Linker ligation (Figure 2)
25. Incubate the ligation mixes for 10 - 16 hours at 16 °C in a thermal cycler (Note 31).
SECTION: 3.4.1 Linker ligation (Figure 2)
26. Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation mix to stop the ligation reaction.
SECTION: 3.4.2 RNA fragmentation (Figure 2)
27. Perform fragmentation calibration test (see Box 2)
SECTION: 3.4.2 RNA fragmentation (Figure 2)
28. Add 20 µl of 2× alkaline fragmentation solution to each sample (from the ligation reaction) and mix. Do not perform fragmentation steps for the oGAB11 ligation control.
SECTION: 3.4.2 RNA fragmentation (Figure 2)
29. Incubate the sample at 95 °C in a thermal cycler for the calibrated time (see Box 2) to fragment RNA (see Note 32).
SECTION: 3.4.2 RNA fragmentation (Figure 2)
30. To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
31. Prepare RNA ladder and un-ligated oGAB11 control by adding 1.0 µl of RNA control ladder or oGAB11 to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and oGAB11 control, mix well (see Note 34).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
32. Prepare all RNA samples (including the ligated oGAB11 control) by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
33. Denature the RNA sample, RNA control samples (ligated oGAB11 and un-ligated oGAB11 controls), and RNA ladder for 2 min at 80 °C. Place the samples on ice.
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
34. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
35. Load gel with the RNA ladder, RNA control samples (ligated oGAB11 and un-ligated oGAB11), and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
36. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
37. Visualize the gel under blue/UV light.
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
38. For the fragmented RNA sample, excise region between 40 and 110 nt. For the ligated oGAB11, excise a narrow band ~55 nt (see Note 36).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
39. Perform rapid gel extraction on the excised gel (see Box 3).
SECTION: 3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
40. After rapid gel extraction is performed, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0) (at this point, RNA can be stored up to 3 months at -80 °C). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).
SECTION: 1.2.1 Washing and counting cells
None. The following steps are for suspension cells, if working with adherent cells see Note 22 in the Guidelines section for steps on how to prepare the cells.
SECTION: 1.2.1 Washing and counting cells
None. Count out 1×107 cells as input for each fractionation, including a western blot control sample (see Note 23 in the Guidelines section). If cell numbers are limited, fractionation can be performed on fewer cells (see Note 24 in the Guidelines section).
SECTION: 1.2.1 Washing and counting cells
None. Collect cells by centrifugation at 500g for 2 min at room temperature.
SECTION: 1.2.1 Washing and counting cells
None. Wash with 10 ml of PBS (room temperature).
SECTION: 1.2.1 Washing and counting cells
None. Collect cells by centrifugation at 500g for 2 min at room temperature.
SECTION: 1.2.1 Washing and counting cells
None. Add spike-in cells (Drosophila S2, used for normalization) to the sample cells collected by centrifugation (the addition of a spike-in is optional, see Note 25 in the Guidelines section).
SECTION: 1.2.1 Washing and counting cells
None. Repeat wash (step 8) and centrifugation (step 9).
SECTION: 1.2.1 Washing and counting cells
None. Remove supernatant by aspiration. It is important to remove the supernatant completely to avoid diluting the lysis buffer (see Note 26 in the Guidelines section).
SECTION: 1.3.1 Isolation of nuclei fraction
None. Using a cut P1000 pipette tip, add 200 µl of CLB to cells and mix by pipetting the sample up and down ten times. Transfer sample to an RNase-free 1.5 ml microcentrifuge tube using a cut 1,000 µl pipette tip.
SECTION: 1.3.1 Isolation of nuclei fraction
None. Incubate the cell lysate on ice for 5 min.
SECTION: 1.3.1 Isolation of nuclei fraction
None. During incubation period, pipette 500 µl of SB to an RNase-free 1.5 ml microcentrifuge tube.
SECTION: 1.3.1 Isolation of nuclei fraction
None. After incubation period, use a cut 1,000 µl pipette tip to layer the cell lysate sample onto the SB (see Note 27 in the Guidelines section).
SECTION: 1.3.1 Isolation of nuclei fraction
None. Centrifuge layered lysate at 16,000g for 10 min at 4 °C to collect nuclei.
SECTION: 1.3.1 Isolation of nuclei fraction
None. Pipette off supernatant completely from the nuclei pellet (supernatant contains the cytoplasmic fraction, retain for western blot, described in Box 1)
SECTION: 1.3.1 Isolation of nuclei fraction
None. Wash nuclei pellet with 800 µl of NWB (either by inverting the tube or pipetting up and down using a cut pipette tip).
SECTION: 1.3.1 Isolation of nuclei fraction
None. Collect cell nuclei by centrifugation at 1,150g for 1 min at 4 °C.
SECTION: 1.3.1 Isolation of nuclei fraction
None. Pipette off supernatant from the nuclei pellet. It is important to remove the supernatant completely in order to remove cytoplasmic mature RNAs (see Note 26 in the Guidelines section).
SECTION: 1.3.2 Isolation of chromatin fraction
None. Add 200 µl of GB to the nuclei. Use a cut 1,000 µl tip to resuspend the washed nuclei pellet by pipetting up and down.
SECTION: 1.3.2 Isolation of chromatin fraction
None. Transfer the nuclei suspension to a new 1.5 ml RNase-free microcentrifuge tube.
SECTION: 1.3.2 Isolation of chromatin fraction
None. Layer 200 µl of NLB onto nuclei suspension and briefly mix by pulsed vortexing.
SECTION: 1.3.2 Isolation of chromatin fraction
None. Incubate the mixture on ice for 2 min.
SECTION: 1.3.2 Isolation of chromatin fraction
None. Centrifuge at 18,500g for 2 min at 4 °C to collect the chromatin pellet.
SECTION: 1.3.2 Isolation of chromatin fraction
None. Pipette off supernatant from the chromatin pellet. It is important to completely remove the supernatant in order to remove nucleoplasmic RNAs (retain supernatant for the nucleoplasm fraction of western blot, described in Box 1).
SECTION: 1.3.2 Isolation of chromatin fraction
None. Add 50 µl CRS to the chromatin pellet, transfer pellet in CRS to a new 1.5 ml tube and then resuspend pellet in CRS (see Note 28 in the Guidelines section). Perform this step on the experimental sample (for the preparation of nascent RNA, section 1.3.3) and the control sample (for the western blot analysis, as described in Box 1).
SECTION: 1.3.3 Preparation of nascent RNA
None. Add 700 µl of QIAzol (lysis reagent in miRNeasy mini kit, Qiagen) to the resuspended chromatin (see Note 10 in the Guidelines section re QIAzol).
SECTION: 1.3.3 Preparation of nascent RNA
None. Solubilize chromatin pellet by slowly pipetting up and down using a 1-ml syringe with a 22G needle (see Note 29 in the Guidelines section). Mix very carefully (to avoid spilling the sample) until the solution is homogeneous (at this point the sample can be stored at −80 °C for months).
SECTION: 1.3.3 Preparation of nascent RNA
None. Prepare RNA using the miRNeasy mini kit (or miRNeasy micro kit for small cell numbers, see Materials section 1.3.3, Preparation of nascent RNA, step 2) according to the manufacturer’s instructions, including optional DNase treatment.
SECTION: 1.3.3 Preparation of nascent RNA
None. Measure the prepared RNA quantity and quality using a NanoDrop (see Note 30 in the Guidelines section). The RNA yield is usually in the range of 20–30 µg (this can vary quite a lot depending on the type of cells used). If fractionation was performed on less than 1×107 cells (or RNA yield is less than 20 µg) see Note 24 in the Guidelines section re RNA amount needed to construct sequencing library. Store the isolated RNA at −80 °C (can be stored for months) until ready to construct sequencing library.
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Prepare linker ligation mix for each RNA sample and for oGAB11 in 0.2-ml RNase-free PCR tubes (see Materials section 1.4.1, Linker ligation, step 4). Mix until reaction is homogeneous, poor mixing will negatively affect the ligation efficiency.
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Incubate the ligation samples overnight for 10 - 16 hours at 16 °C in a thermal cycler (Note 31 in the Guidelines section).
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation sample to stop the ligation reaction.
SECTION: 1.4.2 RNA fragmentation (Figure 2)
None. Perform fragmentation calibration test (see Box 2)
SECTION: 1.4.2 RNA fragmentation (Figure 2)
None. Add 20 µl of 2× alkaline fragmentation solution (see Materials section 1.4.2, RNA fragmentation, step 1) to each sample (from the ligation reaction) and mix. Do not perform fragmentation steps for the oGAB11 ligation control.
SECTION: 1.4.2 RNA fragmentation (Figure 2)
None. Incubate the sample at 95 °C in a thermal cycler for the calibrated time (see Box 2) to fragment RNA (see Note 32 in the Guidelines section).
SECTION: 1.4.2 RNA fragmentation (Figure 2)
None. To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33 in the Guidelines section).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Prepare RNA ladder and un-ligated oGAB11 control by adding 1.0 µl of RNA control ladder or oGAB11 to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and oGAB11 control, mix well (see Note 34 in the Guidelines section).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Prepare all RNA samples (including the ligated oGAB11 control) by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Denature the RNA sample, RNA control samples (ligated oGAB11 and un-ligated oGAB11 controls), and RNA ladder for 2 min at 80 °C. Place the samples on ice.
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35 in the Guidelines section).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Load gel with the RNA ladder, RNA control samples (ligated oGAB11 and un-ligated oGAB11), and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Visualize the gel under blue/UV light.
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. For the fragmented RNA sample, excise region between 40 and 110 nt. For the ligated oGAB11, excise a narrow band ~55 nt (see Note 36 in the Guidelines section).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. Perform rapid gel extraction on the excised gel (see Box 3).
SECTION: 1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)
None. After rapid gel extraction is performed, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0) (at this point, RNA can be stored up to 3 months at -80 °C). Alternatively, RNA can be prepared by RNA precipitation (see Note 33 in the Guidelines section).
SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Add 4.6 µl of RT reaction mix to 10 µl of the RNA sample and the oGAB11 control sample.
SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Denature the sample mixtures (including the oGAB11 control) by incubating for 2 min at 80 °C in a thermal cycler. Place on ice.
SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Add 1.3 µl of SUPERase.In/DTT mix (see Materials section 1.4.4, Reverse Transcription, step 4) and mix well.
SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Add 0.8 µl of SuperScript III (200 U/µl) and mix.
SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Incubate for 30 min at 48 °C in a thermal cycler.
SECTION: 1.4.5 Degradation of RNA after cDNA production (Figure 3)
None. Add 1.8 µl of 1 N NaOH, mix well and incubate 20 min at 98 °C. This step degrades the RNA after cDNA was produced by RT.
SECTION: 1.4.5 Degradation of RNA after cDNA production (Figure 3)
None. Neutralize the reaction by adding 1.8 µl of 1 N HCl; mix well and place on ice.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Prepare the DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample buffer and mix.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Add 20 µl of 2× TBU denaturing sample buffer to the cDNA sample and the oGAB11 cDNA control, mix well.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Denature the cDNA sample, oGAB11 cDNA control and DNA control ladder for 3 min at 95 °C in a Thermomixer.  Place the samples on ice.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Prerun a 10% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35 in the Guidelines section).
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Load gel (20 uL per lane, each sample will be split into two lanes) with the cDNA sample, the oGAB11 cDNA control and the DNA ladder. Separate by PAGE at 200 V for 65 min.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Visualize the gel under blue/UV light.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. For the cDNA sample, excise region between ~85 and 160 nt (use oGAB11 as baseline and cut up ~70 nt). For the cDNA oGAB11 control, excise a narrow band ~85 nt. If no cDNA smear is observed see Note 37 in the Guidelines section.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. Perform rapid gel extraction on the excised gel (see Box 3). There will be two gels for one sample since each sample was split into two lanes, combine the two gels from one sample into a single tube.
SECTION: 1.4.6 Size select cDNA and gel extraction (Figure 3)
None. After rapid gel extraction is performed, use the Oligo kit (Zymo) and elute cDNA from column with 15 uL of 10 mM Tris-HCl (pH 8.0) (at this point, the cDNA can be stored indefinitely at −20 °C). Alternatively, cDNA can be prepared by precipitation (see Note 33 in the Guidelines section).
SECTION: 1.4.7 cDNA Circularization (Figure 4)
None. In a 0.2-ml RNase-free PCR tube, add 4 µl of circularization mix to 15 µl of the cDNA sample and the oGAB11 cDNA control sample, mix well.
SECTION: 1.4.7 cDNA Circularization (Figure 4)
None. Add 1 µl of CircLigase (100 U/µl) and mix.
SECTION: 1.4.7 cDNA Circularization (Figure 4)
None. Incubate the CircLigase reaction for 60 min at 60 °C and then 10 min at 80 °C in a thermal cycler. Circularized cDNA can be stored indefinitely at −20 °C.
SECTION: 1.4.7 cDNA Circularization (Figure 4)
None. Optional: at this point a specific depletion of highly abundant mature RNAs can be performed (see Note 38 in the Guidelines section)
SECTION: 1.4.8 PCR amplification (Figure 4)
None. Perform PCR amplification test (Box 4)
SECTION: 1.4.8 PCR amplification (Figure 4)
None. Prepare PCR mix for four PCR amplification reactions for both the cDNA sample and the oGAB11 control cDNA sample (see Materials section 1.4.8, PCR amplification, step 4). Mix well and store on ice.
SECTION: 1.4.8 PCR amplification (Figure 4)
None. For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
SECTION: 1.4.8 PCR amplification (Figure 4)
None. Add 1 µl of circularized cDNA and mix it well.
SECTION: 1.4.8 PCR amplification (Figure 4)
None. Perform PCR amplifications (as shown in Table 2) with determined optimal number of PCR cycles (see Box 4).

 
 TABLE 2 | PCR amplification
    Cycle numberDenatureAnnealExtend198 °C, 30 s  2–1498 °C, 10 s60 °C, 10 s72 °C, 5 s

 
 
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. Prepare DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of DNase-free H2O. Add 2 µl of 6× DNA loading dye and mix well.
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. Add 3.4 µl of 6× DNA loading dye to each PCR sample tube and mix well.
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. Load gel with the PCR products and DNA control ladder. Separate by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. Visualize the gel under blue/UV light.
SECTION: 1.4.9 PCR size selection and gel extraction (Figure 4)
None. For each sample, excise the band from the gel that contains the PCR product. The NET-seq library is a broad band that runs at ~150 nt (use oGAB as baseline and cut up ~50 nt). Avoid contamination from the lower band that runs at ~120 nt, representing PCR product from empty circles (see Note 39). The oGAB11 control sample is not subjected to this step, or any further steps.
SECTION: 1.4.10 PCR product cleanup
None. Perform the first four steps of rapid gel extraction (see Box 3)
SECTION: 1.4.10 PCR product cleanup
None. After centrifugation, add 670 µl of DNA soaking buffer and mix.
SECTION: 1.4.10 PCR product cleanup
None. Incubate the samples overnight at room temperature in a Thermomixer (set at 1,500 r.p.m.).
SECTION: 1.4.10 PCR product cleanup
None. Use a cut 1,000 µl pipette tip to transfer the gel slurry into a microcentrifuge tube filter.
SECTION: 1.4.10 PCR product cleanup
None. Centrifuge at 20,000g for 3 min at room temperature. Transfer the eluate to a new 1.5 ml microcentrifuge tube.
SECTION: 1.4.10 PCR product cleanup
None. Precipitate the NET-seq library by adding 2 µl of 15 mg/ml GlycoBlue and 680 µl of isopropanol. Mix well then incubate the precipitations at −20 °C for ≥1 h (the DNA precipitations can be stored at −20 °C overnight).
SECTION: 1.4.10 PCR product cleanup
None. Centrifuge at 20,000g for 30 min at 4 °C to pellet the RNA.
SECTION: 1.4.10 PCR product cleanup
None. Remove the supernatant and wash the pellet with 750 µl of 80% (vol/vol) ice-cold ethanol.
SECTION: 1.4.10 PCR product cleanup
None. Spin the sample at 20,000g for 2 min at 4 °C, and then remove the supernatant. Air-dry the RNA pellet for 10 min at room temperature.
SECTION: 1.4.10 PCR product cleanup
None. Resuspend the NET-seq library in 7 - 10 µl of Tris-HCl (10 mM, pH 8.0). The DNA sequencing library can be stored indefinitely at −20 °C.
SECTION: 1.4.11 Quantification and characterization of NET-seq library (Figure 4)
None. Prepare a 1:5 dilution of the NET-seq library by adding 1 µl of the NET-seq library to 4 µl of Tris-HCl (10 mM, pH 8.0); mix well (see Note 40 in the Guidelines section).
SECTION: 1.4.11 Quantification and characterization of NET-seq library (Figure 4)
None. Use 1 µl of the diluted NET-seq library for quantification with the Qubit fluorometer using the Qubit dsDNA HS assay kit. Prepare the sample and perform the measurement according to the manufacturer’s protocol (see Note 41 in the Guidelines section).
SECTION: 1.4.11 Quantification and characterization of NET-seq library (Figure 4)
None. Use 1 µl of the diluted NET-seq library for characterization on the Agilent Bioanalyzer; use the high-sensitivity DNA analysis kit according to the manufacturer’s instructions.
SECTION: 1.4.11 Quantification and characterization of NET-seq library (Figure 4)
None. Sequence using oLSC006. Sequence the human NET-seq library from the 3′ end (75 bp single-end sequencing) on the Illumina platform using oLSC006 (Table 1 in the Materials section) as a custom sequencing primer. Perform sequencing according to the manufacturer’s instructions. NET-seq libraries are typically sequenced on MiSeq, HiSeq or NextSeq next-generation sequencing platforms. A reasonable coverage is obtained with 100–200 million reads.
SECTION: 1.5.1 Alignment of sequencing reads
None. Assess read quality control with FastQC.
SECTION: 1.5.1 Alignment of sequencing reads
None. Displace 5’ end random barcode sequence from sequence to read identifier line and save as a separate fastq file. This barcode serves as a unique molecular identifier (UMI) and originates from the linker (see Table 1 in the Materials section).
SECTION: 1.5.1 Alignment of sequencing reads
None. Align fastq files with and without UMI sequences separately to a human reference genome using STAR (See Note 43 in the Guidelines section). In case a Drosophila spike-in was used, concatenate the human and Drosophila fasta files to generate a combined reference genome.
SECTION: 1.5.2 Filter out read alignments that do not reflect RNA polymerase position.
None. Remove alignments resulting from RT mispriming events, i.e. cases where RT priming did not occur at the complementary sequence site on the ligated linker but instead at a position within the RNA fragment. The resulting reads can be identified as they lack the random UMI sequence encoded on the linker and therefore align well to the reference genome both with and without UMI displacement: when the STAR alignment score of a read with the UMI sequence equals the UMI length (10nt) plus the alignment score from that read without UMI.
SECTION: 1.5.2 Filter out read alignments that do not reflect RNA polymerase position.
None. Remove alignments that map to multiple genomic locations by filtering for their MAPQ value (< 50) using samtools.
SECTION: 1.5.2 Filter out read alignments that do not reflect RNA polymerase position.
None. Remove alignments resulting from PCR duplication events, i.e. cases of multiple amplicons that originate from the same RNA fragment. The resulting multiple reads are bioinformatically indistinguishable as their alignments share the same position, strand, chromosome, UMI sequence and CIGAR score. To filter out these PCR duplicates, we maintain only one of the alignments.
SECTION: 1.5.2 Filter out read alignments that do not reflect RNA polymerase position.
None. Remove alignments that could arise from splicing intermediates, i.e. 3’ ends of annotated exons and introns.
SECTION: 1.5.3 Determine RNA polymerase coverage genome-wide from read alignments.
None. Use HTseq to generate a coverage (bedgraph) file containing the read counts at a base-pair resolution over the reference genome as determined from the 5’ end of the read alignments (without UMI) on the opposite strand. The resulting positions correspond to the 3’ ends of nascent RNA and thereby map RNA polymerase positions. The coverage files will be used for a variety of downstream bioinformatics analyses such as visual inspection (e.g. using IGV), metagene profiling (e.g. Bedtools), differential expression (e.g. DEseq2).
SECTION: Box 1 | Western blot control: checking cell fractionation
None. The western blot control is used to determine whether or not the cellular fractionation protocol has been executed successfully. The efficiency of the fractionation can be quantified by measuring the amount of elongating Pol II captured in the chromatin fraction (ideally  ≥95%). When the fractionation protocol is being performed for the first time or being tested on a new cell type, the cytoplasm, nucleoplasm, and chromatin fractions should be checked for subcellular marker proteins (see Note 5 in the Guidelines section) to confirm the fractionation is working (see Note 20 in the Guidelines section).
SECTION: Box 1 | Western blot control: checking cell fractionation
None. Adjust the nucleoplasmic (from steps 1.3.2) and chromatin fraction (from steps 1.3.2) volumes to the cytoplasmic fraction (from steps 1.3.1) volume by adding 1× PBS. Mix each fraction well and ensure the chromatin pellet is completely resuspended (see Note 7 in the Guidelines section).
SECTION: Box 1 | Western blot control: checking cell fractionation
None. Add 50 µl of 2× SDS buffer (see Materials section 1.3.2, Isolation of chromatin fraction, step 7) to 50 µl of each subcellular fraction and boil the samples at 95 °C for 5 min (at this point, samples can be stored for months at −20 °C).
SECTION: Box 1 | Western blot control: checking cell fractionation
None. Separate samples by standard SDS-PAGE (loading 10 µl of each boiled sample per lane is usually sufficient).
SECTION: Box 1 | Western blot control: checking cell fractionation
None. Probe the membrane overnight at 4 °C with Pol II CTD Ser2-P antibody (3E10, 1:1,000 dilution) and Pol II CTD Ser5-P antibody (3E8, 1:1,000 dilution). These antibodies target transcribing RNA Pol II (see Note 6 in the Guidelines section).
SECTION: Box 2 | Fragmentation test
None. Take 9 samples of RNA (~1ug each) from a total RNA sample (or any other easily obtained RNA sample). Re-suspend each RNA sample in 8uL of RNase-free water and add 12uL of the linker ligation mix, excluding the linker and ligase (see Materials section 1.4.1, Linker ligation, step 4). Add 20 µl of 2× alkaline fragmentation solution (see Materials section 1.4.2, RNA fragmentation, step 1) to each sample and mix well (see Note 32 in the Guidelines section); fragmentation is not performed for the oGAB11 control.
SECTION: Box 2 | Fragmentation test
None. Fragment each RNA sample at 95 °C in a thermal cycler for the following times: 0, 15, 20, 25, 30, 35, 40, 45, and 50 minutes.
SECTION: Box 2 | Fragmentation test
None. To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33 in the Guidelines section).
SECTION: Box 2 | Fragmentation test
None. Prepare RNA ladder (denoted as L in figure) by adding 1.0 µl of RNA control ladder to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and mix well.
SECTION: Box 2 | Fragmentation test
None. Prepare all RNA samples by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
SECTION: Box 2 | Fragmentation test
None. Denature the RNA sample and RNA ladder for 2 min at 80 °C. Place the samples on ice.
SECTION: Box 2 | Fragmentation test
None. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35 in the Guidelines section).
SECTION: Box 2 | Fragmentation test
None. Load gel with the RNA ladder and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
SECTION: Box 2 | Fragmentation test
None. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: Box 2 | Fragmentation test
None. Visualize the gel under blue/UV light. The optimal fragmentation time is when most RNA molecules are in the required size range, between 40 and 110 nt (the optimal fragmentation time in the figure below is between 25 and 30 minutes).
SECTION: Box 3 | Rapid gel extraction
None. Use a 21G needle to pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube.
SECTION: Box 3 | Rapid gel extraction
None. Place the pierced 0.5-ml tube into a 1.5-ml RNase-free microcentrifuge tube.
SECTION: Box 3 | Rapid gel extraction
None. Put the gel slice (from fragmentation, RT, or PCR) into the pierced 0.5-ml tube.
SECTION: Box 3 | Rapid gel extraction
None. Centrifuge the nestled tubes, containing the gel slice, at 20,000g for 4 min at room temperature.
SECTION: Box 3 | Rapid gel extraction
None. Add 200 µl of RNase-free H2O to the chopped-up gel and mix.
SECTION: Box 3 | Rapid gel extraction
None. Incubate the sample mixture for 10 min at 70 °C in a Thermomixer.
SECTION: Box 3 | Rapid gel extraction
None. Vortex the mixture for 30 s at medium intensity.
SECTION: Box 3 | Rapid gel extraction
None. Cut the tip off of a 1,000-µl pipette tip and transfer the gel mixture into a microcentrifuge tube filter.
SECTION: Box 3 | Rapid gel extraction
None. Centrifuge the mixture at 20,000g for 3 min at room temperature.
SECTION: Box 4 | PCR amplification test
None. Prepare the PCR master mix (see Materials section 1.4.8, PCR amplification, step 4) for four pilot PCR amplifications for each cDNA sample and the oGAB11 cDNA control sample (denoted as O in figure). Mix well and store it on ice.
SECTION: Box 4 | PCR amplification test
None. For each PCR sample, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
SECTION: Box 4 | PCR amplification test
None. Add 1 µl of circularized cDNA and mix it well.
SECTION: Box 4 | PCR amplification test
None. Perform PCR pilot amplifications (described in Table 2). Remove one PCR tube for each sample at the end of the extension step after 6, 8, 10 and 12 amplification cycles.
SECTION: Box 4 | PCR amplification test
None. Add 3.4 µl of 6× DNA loading dye to each tube and mix well.
SECTION: Box 4 | PCR amplification test
None. Prepare DNA control ladder (denoted as L in figure) by adding 1.0 µl of ladder to 9 µl of DNase-free H2O. Then add 2 µl of 6× DNA loading dye and mix well.
SECTION: Box 4 | PCR amplification test
None. Separate the PCR products and the DNA control ladder by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.
SECTION: Box 4 | PCR amplification test
None. Stain the gel in 50 ml of gel staining solution (see Materials section 1.4.8, PCR amplification) for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).
SECTION: Box 4 | PCR amplification test
None. Visualize the gel under blue/UV light and identify the optimal PCR amplification cycles for each cDNA sequencing library. The optimal PCR amplification cycles for the NET-seq library, which runs at ~150 nt, is determined by the presence of a clear band at ~150 nt and no higher-molecular-weight PCR products (the optimal number of cycles in the figure above is between 6 and 8).
SECTION: 1.4 Constructing DNA sequencing library from isolated nascent RNA (Figure 1b)
None.   

SECTION: Box 2 | Fragmentation test
None.   

SECTION: Box 3 | Rapid gel extraction
None.   

SECTION: Box 4 | PCR amplification test
None.   

SECTION: 1.4.4 Reverse transcription (Figure 3)
None. Prepare RT reaction mix in a 0.2-ml RNase-free PCR tube (see Materials section 1.4.4, Reverse Transcription, step 3), store on ice.
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Denature the RNA sample (from the cellular fractionation, section 1.3.3) and 5 µl of the oGAB11 control (10 µM; Table 1 in the Materials section) for 2 min at 80 °C in a Thermomixer. Place on ice.
SECTION: 1.4.7 cDNA Circularization (Figure 4)
None. Prepare circularization mix (see Materials section 1.4.7, Circularization of cDNA, step 2) and store it on ice.
SECTION: 1.5 Bioinformatics analysis (Figure 5)
None. Below we describe in brief the custom bioinformatics pipeline (see Figure 5) that is used to generate NET-seq coverage files. The scripts are available at Churchman lab GitHub . See Note 42 in the Guidelines section for a description of the improvements relative to our previous pipeline (Mayer et al. 2015).
SECTION: Box 2 | Fragmentation test
None. Performing RNA fragmentation results in a homogeneous RNA pool which helps to avoid length biases in any downstream enzymatic reactions. The fragmentation test can be performed using total RNA (or any easily-obtained RNA sample). Once the appropriate fragmentation time is determined (where most of the RNA molecules range from 40 –110 nt), fragmentation can be performed on the nascent RNA (obtained from cellular fractionation) using the determined time (see directly above); the size range of the nascent RNA and total RNA pools do not vary enough for the fragmentation time to be different.
SECTION: Box 3 | Rapid gel extraction
None. This protocol for rapidly extracting RNA or cDNA from a polyacrylamide TBE-urea gel is similar to the gel extraction protocol described by the Weissman laboratory (Ingolia et al. 2012; Churchman and Weissman 2012).
SECTION: Box 4 | PCR amplification test
None. The NET-seq library is PCR amplified using an Illumina index forward primer (Barcode primer, see Table 1 in the Materials section) and a reverse primer (oNTI231; Table 1 in the Materials section) specific to NET-seq (Figure 4). For each NET-seq library preparation, the minimum number of PCR cycles needed to obtain a sequencing library must to be determined. The NET-seq library is then amplified with the determined minimal amount of PCR cycles; this avoids overamplification which can result in unwanted products such as PCR duplicates.
SECTION: 1.4.1 Linker ligation (Figure 2)
None. Spray down all work surfaces, pipettes, etc. with RNase away
SECTION: 1.3 Cellular Fractionation (Figure 1a)
None.   

SECTION: 1.4.1 Linker ligation (Figure 2)
None.   

SECTION: 1.4.4 Reverse transcription (Figure 3)
None.   

SECTION: 1.4.7 cDNA Circularization (Figure 4)
None.   

SECTION: 1.5 Bioinformatics analysis (Figure 5)
None.    

SECTION: Preparation of nascent RNA
2. 1. Add 700 μl of QIAzol lysis reagent (part of miRNeasy mini kit, Qiagen) to the resuspended chromatin
2.  Mix thoroughly by slowly pipetting up and down using a 1-ml syringe with a 22G needle. Alternatively, the chromatin pellet can also be solubilized by gentle vortexing. Mix very carefully until the solution is homogeneous. Mix it slowly to avoid spilling the sample. (at this point the sample can be stored at −80 °C for months).
3. Prepare RNA using the miRNeasy mini kit according to the manufacturer’s instructions, including the optional on-column DNase treatment using the RNase-free DNase set (Qiagen).
4. Elute the nascent RNA in 25 µl RNase-free H2O. Assess the quantity and quality of the prepared RNA using NanoDrop 2000 (Thermo Scientific). RNA yield from one whole liver typically ranged from 10 to 56 µg. The absorbance A260/A280 ratios were around 2.1.
SECTION: Barcode DNA linker ligation
3. 1. Spray down all work surfaces, pipettes, etc. with RNase away
2. Denature the RNA sample (from the cellular fractionation, section 1.3.3) and 5 µl of the oGAB11 control (10 µM; Table 1 in the Materials section) for 2 min at 80 °C in a Thermomixer. Place on ice.
3. Prepare linker ligation mix for each RNA sample and for oGAB11 in 0.2-ml RNase-free PCR tubes (see Materials section 1.4.1, Linker ligation, step 4). Mix until reaction is homogeneous, poor mixing will negatively affect the ligation efficiency.
4. Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.
5. Incubate the ligation samples overnight for 10 - 16 hours at 16 °C in a thermal cycler (Note 31 in the Guidelines section).
6. Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation sample to stop the ligation reaction.
 
     Component      Amount per   reaction (µl)      Final        RNA sample      oGAB11 control        PEG8000 (50% v/v)      8.0      8.0      20% (v/v)        DMSO      2.0      2.0      10 % (v/v)        T4 RNA ligase buffer (10x)      2.0      2.0      1x        Barcode DNA linker (1 µg)      1.0      1.0               RNA sample (1 µg)      6.0      -               oGAB11 (10 µM)      -      1.0      0.5 µM        RNase-free H2O      -      5.0               Truncated T4 RNA ligase 2      1.0      1.0      200 U   
 
SECTION: RNA fragmentation
4. Prior to RNA fragmentation, precipitate the RNA to remove PEG, which can affect the fragmentation reaction. 
1. To each sample, add 60 µl of 3 M sodium acetate, 2 µl GlycoBlue (15 mg/ml) and 600 µl of 100 % ethanol.
2. ncubate the samples at -80°C for 45 min. (precipitates can be stored at -80 °C overnight
1. Centrifuge the samples for 30 min at full speed.
2. Wash the samples twice with 500 µl of each 100 % ethanol and 70 % ethanol.
3. Air-dry the pellets for 5 min and resuspend in 20 µl sterile water (10 µl for oGAB11 ligation control). 
SECTION: RNA fragmentation
5. Following precipitation, add 20 µl of 2× alkaline fragmentation solution to each sample and mix. Please note that Steps 4.3 and 5.1 are not performed for the oGAB11 ligation control.
1. Fragment the RNA at 95 °C in a thermal cycler for 20 min. The fragmentation time needs to be adjusted whenever a new batch of alkaline fragmentation solution is applied. An over-fragmentation or under-fragmentation of the nascent RNA pool can lead to systematic biases. In a typical experiment RNA is fragmented between 10 and 40 min at 95 °C. The optimal fragmentation time is when most RNA molecules are in the required size range, usually between 35 and 100 nt. 
2. Following fragmentation, precipitate the RNA and the oGAB11 ligation control as described above (steps 4.1-4.3).
3. Add 20 µl of 2× TBE-urea denaturing sample buffer to each RNA sample and mix it.
4. Prepare the RNA control ladder and the oGAB11 control. Add 1.0 µl 0.5 μl of RNA control ladder or 1 μl oGAB11 to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample buffer to each control sample and mix it.
5. Denature the RNA sample and RNA control samples (oGAB11 ligation control, ladder and oGAB11) for 2 min at 80°C. Cool the samples on ice for 3 min.
6. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE.
7. Separate the fragmented RNA samples and the RNA control samples (including the oGAB11 ligation control) by PAGE at 200 V for 65 min.
8. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.
9. Visualize the fragmented RNA and the oGAB11 ligation control under blue light. For the fragmented RNA, excise the region between 35 (in the middle of the dye front) and 100 nt. For the oGAB11 ligation control, excise the narrow band at ~55 nt.
10. Extract the RNA from the gel slice by rapid gel extraction:
a. Pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube with a 23G needle. 
b. Put the pierced 0.5-ml tube in a 1.5-ml RNase-free microcentrifuge tube. 
c. Combine and place the gel slices for each sample into the inner pierced 0.5-ml tube. 
d. Centrifuge the mixture at 20,000g for 4 min at room temperature.
e. Add 200 µl RNA recovery buffer (Zymo Research, R1070-1-10) to the excised gel slices.
f. Incubate the sample for 15 min at 70°C in a Thermomixer (1500 rpm). 
g. Vortex the mixture for 30 s at a medium intensity setting. 
h. Cut the tip off of a 1,000-µl pipette tip and transfer the gel slurry into a Zymo-Spin IV Column (Zymo Research, C1007-50).
i. Freeze for 2 min at -80°C and subsequently thaw by placing at 70°C for 1 min.
j. Centrifuge the mixture at 20,000g for 30 secat room temperature. 
k. The expected eluate volume is ~200 µl.
11. Precipitate the RNA by adding 90 µl of 3 M sodium acetate, 2 µl GlycoBlue (15 mg/ml) and 900 µl of 100 % ethanol. Proceed as described above (step 28).
12. Re-suspend the size-selected RNA and the oGAB11 ligation control in 10 µl of pre-cooled RNase-free H2O.
SECTION: cDNA synthesis by reverse transcription
6. 1. Synthesize cDNA using the SuperScript™ III First-Strand Synthesis System (Thermo Fischer) following the manufacturer’s instructions. In brief, add 0.8 µl dNTPs (10 mM) and 0.5 µl of the reverse primer oLSC007 (10 µM, Table 2) to 10 µl of RNA sample and to the oGAB11 control. After incubating at 80°C in a thermal cycler for 2 min and cooling on ice for 3 min, add 10 µl of cDNA synthesis mix consisting of 2 µl of 10x RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT, 1 µl of RNaseOUT™ (40 U/µl) and 1 µl of SuperScript® III RT (200 U/µl) to each sample.
2. Incubate the mixture for 30 min at 48°C in a thermal cycler. 
3. Add 1.8 µl of 1 N NaOH, mix well and incubate the reaction for 20 min at 98°C. 
4. Neutralize the reaction by adding 1.8 µl of 1 N HCl; mix well and put the reaction on ice. 
5. Add 20 µl of 2× TBU denaturing sample buffer to each cDNA sample and the oGAB11 cDNA control, and then mix.
6. Prepare the DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample buffer to the ladder and mix.
7. Denature the cDNA sample, oGAB11 cDNA control and DNA control ladder for 3 min at 95°C in a Thermomixer. Cool the samples on ice for 3 min.
8. Pre-run a 10% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. 
9. Separate the cDNA sample, the oGAB11 cDNA control and the DNA ladder by PAGE at 200 V for 65 min.
10. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.
11. Visualize the gel under blue/UV light and excise the cDNA between 85 and 160 nt.
12. Extract the cDNA from the gel slice by rapid gel extraction (see step 5.10). To 200 µl eluate, add 25 µl of 5 M NaCl and mix.
13. Precipitate the cDNA by adding 2 µl GlycoBlue (15 mg/ml) and 750 µl of 100 % ethanol. Proceed as described above (step 28). Resuspend cDNA and oGAB11 control cDNA in 15 µl of pre-cooled RNase-free H2O each. The cDNA can be stored indefinitely at −20°C.
SECTION: Circularization of cDNA
7. 1. Prepare circularization mix (Table 4) and store it on ice.
2. Add 4 µl of circularization mix to 15 µl of cDNA sample and the oGAB11 cDNA control sample in a 0.2-ml RNase-free PCR tube, and then mix well. 
3. Add 1 µl of CircLigase (100 U/µl) and mix.
 
     Component      Amount per reaction   (µl)      Final        CircLigase reaction buffer (10x)      2.0      1x        ATP (1 mM)      1.0      50 µM        MnCl2 (50 mM)      1.0      2.5 mM   

4. Incubate the CircLigase reaction for 60 min at 60°C and for 10 min at 80°C in a thermal cycler. Circularized cDNA can be stored indefinitely at −20°C. 
SECTION: Specific depletion of highly abundant mature RNAs
8. 1. For depletion of the 20 most abundant chromatin-associated mature RNAs (see Materials, step 11), prepare one specific depletion reaction per sample in 0.2-ml DNase-free PCR tubes. Please note that the oGAB11 control sample is not subjected to the depletion procedure.
2. Perform subtractive hybridization in a thermal cycler (Table 7).
3. Prepare Dynabeads MyOne streptavidin C1 (10 mg/ml) at room temperature. 
a. Re-suspend the beads by gentle vortexing. 
b.Transfer 100 µl of beads per depletion reaction to a DNase-free 1.5-ml microcentrifuge tube. 
c.Place the tube on a magnetic rack for 1 min and withdraw all of the supernatant from the tube. 
d.Remove the tube from the magnetic rack and re-suspend beads in 100 µl of bind/wash buffer. 
e.Repeat this washing procedure (steps 3 and 4) two more times. 
f.Place the tube on a magnetic rack for 1 min and withdraw the supernatant from the tube. 
g.Remove the tube from the magnetic rack and re-suspend the beads in 30 µl of bind/wash buffer. 
h.Transfer 25 µl of the re-suspended and washed beads to a new tube. 
i.Place the tube in a Thermomixer at 37°C to equilibrate for 15–30 min.
4. Transfer 40 µl of depletion reaction directly from the 0.2-ml PCR tube in the thermal cycler (from Step 71) to the washed and equilibrated beads in the Thermomixer. Immediately mix by pipetting. 

Composition of the depletion reactions.
 
     Component      Amount per   reaction (µl)      Final        Circularization reaction (from step 69)      20               Depletion DNA oligo pool      4.0      1 µM        SSC, 20x      4.0      2x        DNase-free H2O      12.0          
 
Parameters for depletion of highly abundant mature RNAs.
 
            Temperature      Time        Denature      99°C      90 s        Annealing      99-37°C in 0.1°C steps      1s (per 0.1°C step)        Final annealing      37°C      15 min   
 
5. Incubate the mixture in the Thermomixer for 15 min at 37°C with mixing at 1,000 rpm.
6. Transfer the tubes from the Thermomixer into a magnetic rack and leave them for 1 min. Transfer the supernatant into a new 1.5-ml microcentrifuge tube. The supernatant needs to be transferred carefully. Any remaining magnetic beads in the supernatant will have a negative impact on subsequent steps.
7. Precipitate the oligo-depleted, circularized cDNA by adding 6 µl of 5 M sodium chloride, 2 µl GlycoBlue (15 mg/ml) and 250 µl of 100 % ethanol. Proceed as described above (step 4.1-4.3). Resuspend the cDNA in 10 µl sterile water. DNA can be stored indefinitely at −20°C.
SECTION: PCR amplification of the cDNA sequencing library
9. 1. Prepare PCR mix for four pilot PCR amplification reactions for both the cDNA sample and the oGAB11 control cDNA sample (Table 8). Mix well and store it on ice.
2. For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
3. Add 1 µl of circularized cDNA and mix it well.
4. Perform PCR pilot amplifications (Table 9). Remove one PCR tube for each sample at the end of the extension step after 6, 8, 10 and 12 cycles.

Composition of PCR amplification reactions.
 
     Component      Amount for 4   reactions (µl)      Final        Phusion HF buffer (5x)      15.2      1x        dNTPs (10 mM)      1.5      0.2 mM        Forward primer (Ilumina index primer, 100 µM)      0.4      0.5 µM        oNTI231 (reverse primer, 100 µM)      0.4      0.5 µM        DNase-free H2O      57.6               Phusion DNA polymerase (2 U/µl)      0.9      1.8 U   

Parameters for PCR amplification.

     Cycle number      Denature      Anneal      Extend        1      98°C, 30s                      2-14      98°C, 10 s      60°C, 10 s      72°C, 5 s   


5. Add 3.4 µl of 6× DNA loading dye to each tube and mix well.
6. Prepare DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of DNase-free H2O. Add 2 µl of 6× DNA loading dye and mix well. 
7. Separate the PCR products and the DNA control ladder by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.
8. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.
9. Visualize the gel under blue/UV light and identify the optimal PCR amplification cycle for each cDNA sequencing library. The NET-seq library runs at ~150 nt. The optimal PCR amplification cycle is characterized by a clear band at ~150 nt and the absence of PCR products at the higher-molecular-weight range.
10. Perform four PCR amplification reactions per sample with the optimal amplification cycle. The oGAB11 control sample is not subjected to amplification. Add 6 µl of 6× DNA loading dye to each sample and mix well. Separate the PCR products by TBE gel electrophoresis on a 4% (wt/vol) low melt agarose gel at 80 V for 2 h. 
11. For each sample, excise the band that contains the PCR product from the gel. Excise the broad band at ~150 nt. Avoid contamination from the lower band that runs at ~120 nt, representing PCR product from empty circles. Empty circles are circularized cDNA molecules that arise from unextended RT primers, and hence they do not contain any information about the original nascent RNA.
12. Purify the final NET-seq library using the Nucleospin™ Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions. Elute in 10 µl of Tris-HCl (10 mM, pH 8.0). The DNA sequencing library can be stored indefinitely at −20°C.
SECTION: Quantification and characterization of the NET-seq library
10. 1. Prepare a 1:5 dilution of the NET-seq library by adding 1 µl of the NET-seq library to 4 µl of Tris-HCl (10 mM, pH 8.0); mix well.
2. Use 1 µl of the diluted NET-seq library for quantification with the Qubit fluorometer using the Qubit dsDNA HS assay kit. Prepare the sample and perform the measurement according to the manufacturer’s protocol.
3. Use 1 μl of the diluted NET-seq library for characterization on the Agilent Bioanalyzer; use the high-sensitivity DNA analysis kit according to the manufacturer’s instructions.
4. Sequence the human NET-seq library from the 3′ end on the Illumina platform using oLSC006 (Table 2) as a custom sequencing primer.