TITLE: Profiling metazoan transcription genome-wide with nucleotide resolution using NET-seq  (native elongating transcript sequencing)
AUTHORS: Danya Martell, Robert Ietswaart, Brendan M. Smalec, Stirling Churchman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. NET-seq applied to human cells has uncovered regions of Pol II pausing at the boundaries of retained exons and convergent antisense transcription near transcription start sites (Mayer et al. 2015). It has also been used to investigate regulators of productive elongation (Winter et al. 2017), and the directionality of promoter regions (Jin et al. 2017). Here, we describe the experimental protocol for metazoan cells that includes a spike-in control enabling normalization across samples. We also report on an improved bioinformatics pipeline for NET-seq. Together, the protocol yields a fast and non-perturbative method to map Pol II transcription genome-wide, revealing complex and global transcriptional events.</div><div class = "text-block"><span style = "font-weight:bold;">1. Introduction</span></div><div class = "text-block">Transcription regulation has many layers of complexity. Canonically, we understand that transcription is controlled by promoters and distal regulatory regions, or enhancers, that are proposed to be the primary determinant of cell type–specific gene expression. Additionally, transcription elongation and other post-initiation events, such as promoter-proximal pausing, are emerging as crucial regulatory steps in controlling gene expression (Adelman and Lis 2012; Margaritis and Holstege 2008). Non-coding RNA transcripts, such as long intergenic non-coding and antisense RNAs, are involved in gene regulation of specific genes or of larger regions, such as in X chromosome inactivation (Rinn and Chang 2012; Ietswaart, Wu, and Dean 2012). </div><div class = "text-block">The deep analysis of steady-state pools of RNA have yielded useful insights into nascent transcription (Boswell et al. 2017), but they fail to capture unstable RNA species, such as enhancer RNAs, and transcriptional pausing. Traditional transcription run-on techniques only observe a few genes at a time. As transcription elongation has emerged as a crucial regulatory step in controlling gene expression, it is critical to directly monitor the elongation process to identify all layers of gene regulation. Native elongating transcript sequencing (NET-seq) is a non-perturbative method which detects actively elongating RNA polymerase II (Pol II) genome-wide --with strand-specific nucleotide resolution, in vivo. This chapter provides a step-by-step protocol for NET-seq and a bioinformatics pipeline for sequence analysis.  </div><div class = "text-block">The NET-seq protocol begins with purifying nascent RNA, which is done by cellular fractionation in metazoan cells (Figure 1a). Briefly, cells are lysed, and the nuclei are isolated from the cytoplasm by centrifugation through a sucrose cushion buffer. The nuclei are then washed to eliminate any remaining cytoplasmic matter. The chromatin fraction is isolated from the nucleoplasm using urea, salt, and mild detergents. While urea removes most chromatin-bound proteins, it does not remove histones or elongating RNA polymerase from DNA. The RNA polymerase-RNA-DNA ternary complex is stable and capable of withstanding high concentrations of salt, urea, and detergents (Wuarin and Schibler 1994; Cai and Luse 1987). Due to this stability, the isolated chromatin fraction is enriched for nascent RNAs that arise from transcriptionally engaged RNA polymerase complexes. As the histone proteins also remain on DNA after urea treatment, the chromatin fraction can be isolated through low-speed centrifugation. Isolating the chromatin fraction completes the process of purifying the nascent RNA. An advantage of this purification approach is that a restart of transcription in vitro is not required and can therefore isolate nascent RNA from Pol II in multiple transcriptional states. Furthermore, possible biases arising from antisera-based purification of Pol II from epitope masking and cross-reactivity are avoided. </div><div class = "text-block"><span>After the nascent RNA has been purified, the sequencing library preparation can proceed (Figure 1b). This begins with the ligation of a linker to the 3′ end of the RNA, which preserves the information on the 3′ end of RNAs and allows for nucleotide precision of the location of Pol II. The linker contains a random sequence at the 5′ end that serves as a unique molecular identifier (UMI), enabling the bioinformatic detection of multiple library generation biases, including PCR duplicates and reverse transcription mispriming events. After 3’ ligation of the linker, the RNA is fragmented and size selected. Fragmenting the RNA helps avoid length biases in any downstream enzymatic reactions. After fragmentation, the RNA is reverse transcribed to create single-stranded cDNA using a primer with a long overhang. The cDNA, containing a 3' adaptor, is then circularized. Sequence elements introduced in the RT primer can then serve as a 5' adaptor to allow for PCR amplification (see Figure 1b). The PCR product is then sequenced using a next generation sequencing platform, typically Illumina. The resulting sequencing reads are aligned, and data analysis is then performed. The NET-seq bioinformatics pipeline for sequence alignment has improved compared to the original pipeline in </span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528962/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Mayer et al. 2015</span></a><span>; the RT mispriming and PCR duplicate removal scripts now perform a more stringent comparison of read alignment (reducing the number of reads filtered out at this stage) which generates a 2-3 fold higher coverage (scripts are available at </span><a href="http://github.com/churchmanlab/MiMB2019NETseq" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Churchman lab GitHub</span></a><span>). </span></div><div class = "text-block">NET-seq surveys transcriptional processes and Pol II occupancy with DNA strand specificity and nucleotide resolution. It does not require any genetic modification or any metabolic labeling of nascent RNA. NET-seq also works on a number of different cell types including various human cell lines (Winter et al. 2017; Mayer et al. 2015), as well as mouse and Drosophila cells (see Note 19). It should be noted that RNA processing intermediates and some mature chromatin-associated RNAs may be included in the library, although these can be computationally removed through comparison with gene annotations. Furthermore, NET-seq does not provide information about the positions of pre-initiation complexes and transcription start sites. In sum, NET-seq is a straightforward, easy to use, in vivo, high resolution methodology that captures complex and global transcriptional events by directly monitoring nascent coding, non-coding, antisense, intergenic, and enhancer RNAs.</div></div>

[STEPS]
?. [2.1 Preparation of buffers and working area for cellular fractionation.]
?. [2.1 Preparation of buffers and working area for cellular fractionation.]
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
Add 20 µl of 2× TBU denaturing sample buffer to the cDNA sample and the oGAB11 cDNA control, mix well.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
Visualize the gel under blue/UV light.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [3.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [3.4.7 cDNA Circularization (Figure 4)]
Prepare circularization mix and store it on ice.
?. [3.4.7 cDNA Circularization (Figure 4)]
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.
?. [3.4.7 cDNA Circularization (Figure 4)]
Add 1 µl of CircLigase (100 U/µl) and mix.
?. [3.4.7 cDNA Circularization (Figure 4)]
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.
?. [3.4.7 cDNA Circularization (Figure 4)]
Optional: at this point a specific depletion of highly abundant mature RNAs can be performed (see Note
?. [3.4.8 PCR amplification (Figure 4)]
Perform PCR amplification test (Box 4)
?. [3.4.8 PCR amplification (Figure 4)]
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.
?. [3.4.8 PCR amplification (Figure 4)]
For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
?. [3.4.8 PCR amplification (Figure 4)]
Add 1 µl of circularized cDNA and mix it well.
?. [3.4.8 PCR amplification (Figure 4)]
Perform PCR amplifications (as shown in Table 2) with determined optimal number of PCR cycles (see Box 4).
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
Add 3.4 µl of 6× DNA loading dye to each PCR sample tube and mix well.
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
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).
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
Visualize the gel under blue/UV light.
?. [3.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [3.4.10 PCR product cleanup]
Perform the first four steps of rapid gel extraction (see Box 3)
?. [3.4.10 PCR product cleanup]
After centrifugation, add 670 µl of DNA soaking buffer and mix.
?. [3.4.10 PCR product cleanup]
Incubate the samples overnight at room temperature in a Thermomixer (set at 1,500 r.p.m.).
?. [3.4.10 PCR product cleanup]
Use a cut 1,000 µl pipette tip to transfer the gel slurry into a microcentrifuge tube filter.
?. [3.4.10 PCR product cleanup]
Centrifuge at 20,000g for 3 min at room temperature. Transfer the eluate to a new 1.5 ml microcentrifuge tube.
?. [3.4.10 PCR product cleanup]
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).
?. [3.4.10 PCR product cleanup]
Centrifuge at 20,000g for 30 min at 4 °C to pellet the RNA.
?. [3.4.10 PCR product cleanup]
Remove the supernatant and wash the pellet with 750 µl of 80% (vol/vol) ice-cold ethanol.
?. [3.4.10 PCR product cleanup]
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.
?. [3.4.10 PCR product cleanup]
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.
?. [3.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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).
?. [3.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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).
?. [3.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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.
?. [3.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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.
?. [3.5 Bioinformatics analysis (Figure 5)]
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).
?. [3.5.1 Alignment of sequencing reads]
Assess read quality control with FastQC.
?. [3.5.1 Alignment of sequencing reads]
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).
?. [3.5.1 Alignment of sequencing reads]
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.
?. [3.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
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.
?. [3.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
Remove alignments that map to multiple genomic locations by filtering for their MAPQ value (
?. [3.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
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.
?. [3.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
Remove alignments that could arise from splicing intermediates, i.e. 3’ ends of annotated exons and introns.
?. [3.5.3 Determine RNA polymerase coverage genome-wide from read alignments.]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
Separate samples by standard SDS-PAGE (loading 10 µl of each boiled sample per lane is usually sufficient).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
Prepare all RNA samples by adding 1.0 µl of 2× TBU denaturing sample buffer to each sample, mix well.
?. [Box 2 | Fragmentation test]
Denature the RNA sample and RNA ladder for 2 min at 80 °C. Place the samples on ice.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
Load gel with the RNA ladder and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
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).
?. [Box 3 | Rapid gel extraction]
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).
?. [Box 3 | Rapid gel extraction]
Use a 21G needle to pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube.
?. [Box 3 | Rapid gel extraction]
Place the pierced 0.5-ml tube into a 1.5-ml RNase-free microcentrifuge tube.
?. [Box 3 | Rapid gel extraction]
Put the gel slice (from fragmentation, RT, or PCR) into the pierced 0.5-ml tube.
?. [Box 3 | Rapid gel extraction]
Centrifuge the nestled tubes, containing the gel slice, at 20,000g for 4 min at room temperature.
?. [Box 3 | Rapid gel extraction]
Add 200 µl of RNase-free H2O to the chopped-up gel and mix.
?. [Box 3 | Rapid gel extraction]
Incubate the sample mixture for 10 min at 70 °C in a Thermomixer.
?. [Box 3 | Rapid gel extraction]
Vortex the mixture for 30 s at medium intensity.
?. [Box 3 | Rapid gel extraction]
Cut the tip off of a 1,000-µl pipette tip and transfer the gel mixture into a microcentrifuge tube filter.
?. [Box 3 | Rapid gel extraction]
Centrifuge the mixture at 20,000g for 3 min at room temperature.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
For each PCR sample, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
?. [Box 4 | PCR amplification test]
Add 1 µl of circularized cDNA and mix it well.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
Add 3.4 µl of 6× DNA loading dye to each tube and mix well.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
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).
?. [Box 4 | PCR amplification test]
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).
?. [2.4.2 RNA fragmentation]
3.1 Isolation of nuclei fraction
?. [1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.]
Spray down all worksurfaces, pipettes, etc. with RNase away.
?. [1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.]
Prepare CLB, SB, NWB, GB, NLB, and CRS buffers (see Materials section 1.1 for buffer components) and store all solutions on ice.
?. [1.1 Preparation of buffers and working area for cellular fractionation. Prepare all buffers on the day of fractionation before collecting cells.]
Make sure tabletop centrifuge is at 4 °C.
?. [1.2 Preparation of cells for fractionation]
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).
?. [3.2.1 Washing and counting cells]
The following steps are for suspension cells, if working with adherent cells see Note 22 for steps on how to prepare the cells.
?. [3.2.1 Washing and counting cells]
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).
?. [3.2.1 Washing and counting cells]
Collect cells by centrifugation at 500g for 2 min at room temperature.
?. [3.2.1 Washing and counting cells]
Wash with 10 ml of PBS (room temperature).
?. [3.2.1 Washing and counting cells]
Collect cells by centrifugation at 500g for 2 min at room temperature.
?. [3.2.1 Washing and counting cells]
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).
?. [3.2.1 Washing and counting cells]
Repeat wash (step 3) and centrifugation (step 4).
?. [3.2.1 Washing and counting cells]
Remove supernatant by aspiration. It is important to remove the supernatant completely to avoid diluting the lysis buffer (see Note 26).
?. [3.3 Cellular Fractionation (Figure 1a)]
?. [3.3.1 Isolation of nuclei fraction]
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.
?. [3.3.1 Isolation of nuclei fraction]
Incubate the cell lysate on ice for 5 min.
?. [3.3.1 Isolation of nuclei fraction]
During incubation period, pipette 500 µl of SB to an RNase-free 1.5 ml microcentrifuge tube.
?. [3.3.1 Isolation of nuclei fraction]
After incubation period, use a cut 1,000 µl pipette tip to layer the cell lysate sample onto the SB (see Note 27).
?. [3.3.1 Isolation of nuclei fraction]
Centrifuge layered lysate at 16,000g for 10 min at 4 °C to collect nuclei.
?. [3.3.1 Isolation of nuclei fraction]
Pipette off supernatant completely from the nuclei pellet (supernatant contains the cytoplasmic fraction, retain for western blot, described in Box 1)
?. [3.3.1 Isolation of nuclei fraction]
Wash nuclei pellet with 800 µl of NWB (either by inverting the tube or pipetting up and down).
?. [3.3.1 Isolation of nuclei fraction]
Collect cell nuclei by centrifugation at 1,150g for 1 min at 4 °C.
?. [3.3.1 Isolation of nuclei fraction]
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).
?. [3.3.2 Isolation of chromatin fraction]
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.
?. [3.3.2 Isolation of chromatin fraction]
Transfer the nuclei suspension to a new 1.5 ml RNase-free microcentrifuge tube.
?. [3.3.2 Isolation of chromatin fraction]
Layer 200 µl of NLB onto nuclei suspension and mix by pulsed vortexing.
?. [3.3.2 Isolation of chromatin fraction]
Incubate the mixture on ice for 2 min.
?. [3.3.2 Isolation of chromatin fraction]
Centrifuge at 18,500g for 2 min at 4 °C to collect the chromatin pellet.
?. [3.3.2 Isolation of chromatin fraction]
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).
?. [3.3.2 Isolation of chromatin fraction]
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).
?. [3.3.3 Preparation of nascent RNA]
Add 700 µl of QIAzol (lysis reagent in miRNeasy mini kit, Qiagen) to the resuspended chromatin (see Note 10 re QIAzol).
?. [3.3.3 Preparation of nascent RNA]
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.
?. [3.3.3 Preparation of nascent RNA]
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.
?. [3.3.3 Preparation of nascent RNA]
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.
?. [3.4 Constructing DNA sequencing library from isolated nascent RNA (Figure 1b)]
Spray down all work surfaces, pipettes, etc. with RNase away
?. [3.4.1 Linker ligation (Figure 2)]
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.
?. [3.4.1 Linker ligation (Figure 2)]
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.
?. [3.4.1 Linker ligation (Figure 2)]
Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.
?. [3.4.1 Linker ligation (Figure 2)]
Incubate the ligation mixes for 10 - 16 hours at 16 °C in a thermal cycler (Note 31).
?. [3.4.1 Linker ligation (Figure 2)]
Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation mix to stop the ligation reaction.
?. [3.4.2 RNA fragmentation (Figure 2)]
Perform fragmentation calibration test (see Box 2)
?. [3.4.2 RNA fragmentation (Figure 2)]
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.
?. [3.4.2 RNA fragmentation (Figure 2)]
Incubate the sample at 95 °C in a thermal cycler for the calibrated time (see Box 2) to fragment RNA (see Note 32).
?. [3.4.2 RNA fragmentation (Figure 2)]
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).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Prepare all RNA samples (including the ligated oGAB11 control) by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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.
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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.
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Visualize the gel under blue/UV light.
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Perform rapid gel extraction on the excised gel (see Box 3).
?. [3.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.2.1 Washing and counting cells]
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.
?. [1.2.1 Washing and counting cells]
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).
?. [1.2.1 Washing and counting cells]
Collect cells by centrifugation at 500g for 2 min at room temperature.
?. [1.2.1 Washing and counting cells]
Wash with 10 ml of PBS (room temperature).
?. [1.2.1 Washing and counting cells]
Collect cells by centrifugation at 500g for 2 min at room temperature.
?. [1.2.1 Washing and counting cells]
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).
?. [1.2.1 Washing and counting cells]
Repeat wash (step 8) and centrifugation (step 9).
?. [1.2.1 Washing and counting cells]
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).
?. [1.3.1 Isolation of nuclei fraction]
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.
?. [1.3.1 Isolation of nuclei fraction]
Incubate the cell lysate on ice for 5 min.
?. [1.3.1 Isolation of nuclei fraction]
During incubation period, pipette 500 µl of SB to an RNase-free 1.5 ml microcentrifuge tube.
?. [1.3.1 Isolation of nuclei fraction]
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).
?. [1.3.1 Isolation of nuclei fraction]
Centrifuge layered lysate at 16,000g for 10 min at 4 °C to collect nuclei.
?. [1.3.1 Isolation of nuclei fraction]
Pipette off supernatant completely from the nuclei pellet (supernatant contains the cytoplasmic fraction, retain for western blot, described in Box 1)
?. [1.3.1 Isolation of nuclei fraction]
Wash nuclei pellet with 800 µl of NWB (either by inverting the tube or pipetting up and down using a cut pipette tip).
?. [1.3.1 Isolation of nuclei fraction]
Collect cell nuclei by centrifugation at 1,150g for 1 min at 4 °C.
?. [1.3.1 Isolation of nuclei fraction]
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).
?. [1.3.2 Isolation of chromatin fraction]
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.
?. [1.3.2 Isolation of chromatin fraction]
Transfer the nuclei suspension to a new 1.5 ml RNase-free microcentrifuge tube.
?. [1.3.2 Isolation of chromatin fraction]
Layer 200 µl of NLB onto nuclei suspension and briefly mix by pulsed vortexing.
?. [1.3.2 Isolation of chromatin fraction]
Incubate the mixture on ice for 2 min.
?. [1.3.2 Isolation of chromatin fraction]
Centrifuge at 18,500g for 2 min at 4 °C to collect the chromatin pellet.
?. [1.3.2 Isolation of chromatin fraction]
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).
?. [1.3.2 Isolation of chromatin fraction]
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).
?. [1.3.3 Preparation of nascent RNA]
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).
?. [1.3.3 Preparation of nascent RNA]
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).
?. [1.3.3 Preparation of nascent RNA]
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.
?. [1.3.3 Preparation of nascent RNA]
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.
?. [1.4.1 Linker ligation (Figure 2)]
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.
?. [1.4.1 Linker ligation (Figure 2)]
Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.
?. [1.4.1 Linker ligation (Figure 2)]
Incubate the ligation samples overnight for 10 - 16 hours at 16 °C in a thermal cycler (Note 31 in the Guidelines section).
?. [1.4.1 Linker ligation (Figure 2)]
Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation sample to stop the ligation reaction.
?. [1.4.2 RNA fragmentation (Figure 2)]
Perform fragmentation calibration test (see Box 2)
?. [1.4.2 RNA fragmentation (Figure 2)]
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.
?. [1.4.2 RNA fragmentation (Figure 2)]
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).
?. [1.4.2 RNA fragmentation (Figure 2)]
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).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Prepare all RNA samples (including the ligated oGAB11 control) by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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.
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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.
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Visualize the gel under blue/UV light.
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
Perform rapid gel extraction on the excised gel (see Box 3).
?. [1.4.3 Size select Fragmented RNA and perform gel extraction (Figure 2)]
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).
?. [1.4.4 Reverse transcription (Figure 3)]
Add 4.6 µl of RT reaction mix to 10 µl of the RNA sample and the oGAB11 control sample.
?. [1.4.4 Reverse transcription (Figure 3)]
Denature the sample mixtures (including the oGAB11 control) by incubating for 2 min at 80 °C in a thermal cycler. Place on ice.
?. [1.4.4 Reverse transcription (Figure 3)]
Add 1.3 µl of SUPERase.In/DTT mix (see Materials section 1.4.4, Reverse Transcription, step 4) and mix well.
?. [1.4.4 Reverse transcription (Figure 3)]
Add 0.8 µl of SuperScript III (200 U/µl) and mix.
?. [1.4.4 Reverse transcription (Figure 3)]
Incubate for 30 min at 48 °C in a thermal cycler.
?. [1.4.5 Degradation of RNA after cDNA production (Figure 3)]
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.
?. [1.4.5 Degradation of RNA after cDNA production (Figure 3)]
Neutralize the reaction by adding 1.8 µl of 1 N HCl; mix well and place on ice.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
Add 20 µl of 2× TBU denaturing sample buffer to the cDNA sample and the oGAB11 cDNA control, mix well.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
Visualize the gel under blue/UV light.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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.
?. [1.4.6 Size select cDNA and gel extraction (Figure 3)]
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).
?. [1.4.7 cDNA Circularization (Figure 4)]
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.
?. [1.4.7 cDNA Circularization (Figure 4)]
Add 1 µl of CircLigase (100 U/µl) and mix.
?. [1.4.7 cDNA Circularization (Figure 4)]
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.
?. [1.4.7 cDNA Circularization (Figure 4)]
Optional: at this point a specific depletion of highly abundant mature RNAs can be performed (see Note 38 in the Guidelines section)
?. [1.4.8 PCR amplification (Figure 4)]
Perform PCR amplification test (Box 4)
?. [1.4.8 PCR amplification (Figure 4)]
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.
?. [1.4.8 PCR amplification (Figure 4)]
For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
?. [1.4.8 PCR amplification (Figure 4)]
Add 1 µl of circularized cDNA and mix it well.
?. [1.4.8 PCR amplification (Figure 4)]
Perform PCR amplifications (as shown in Table 2) with determined optimal number of PCR cycles (see Box 4). TABLE 2 | PCR amplificationCycle numberDenatureAnnealExtend198 °C, 30 s  2–1498 °C, 10 s60 °C, 10 s72 °C, 5 s
Cycle numberDenatureAnnealExtend198 °C, 30 s  2–1498 °C, 10 s60 °C, 10 s72 °C, 5 s
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
Add 3.4 µl of 6× DNA loading dye to each PCR sample tube and mix well.
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
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).
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
Visualize the gel under blue/UV light.
?. [1.4.9 PCR size selection and gel extraction (Figure 4)]
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.
?. [1.4.10 PCR product cleanup]
Perform the first four steps of rapid gel extraction (see Box 3)
?. [1.4.10 PCR product cleanup]
After centrifugation, add 670 µl of DNA soaking buffer and mix.
?. [1.4.10 PCR product cleanup]
Incubate the samples overnight at room temperature in a Thermomixer (set at 1,500 r.p.m.).
?. [1.4.10 PCR product cleanup]
Use a cut 1,000 µl pipette tip to transfer the gel slurry into a microcentrifuge tube filter.
?. [1.4.10 PCR product cleanup]
Centrifuge at 20,000g for 3 min at room temperature. Transfer the eluate to a new 1.5 ml microcentrifuge tube.
?. [1.4.10 PCR product cleanup]
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).
?. [1.4.10 PCR product cleanup]
Centrifuge at 20,000g for 30 min at 4 °C to pellet the RNA.
?. [1.4.10 PCR product cleanup]
Remove the supernatant and wash the pellet with 750 µl of 80% (vol/vol) ice-cold ethanol.
?. [1.4.10 PCR product cleanup]
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.
?. [1.4.10 PCR product cleanup]
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.
?. [1.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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).
?. [1.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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).
?. [1.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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.
?. [1.4.11 Quantification and characterization of NET-seq library (Figure 4)]
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.
?. [1.5.1 Alignment of sequencing reads]
Assess read quality control with FastQC.
?. [1.5.1 Alignment of sequencing reads]
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).
?. [1.5.1 Alignment of sequencing reads]
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.
?. [1.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
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.
?. [1.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
Remove alignments that map to multiple genomic locations by filtering for their MAPQ value (
?. [1.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
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.
?. [1.5.2 Filter out read alignments that do not reflect RNA polymerase position.]
Remove alignments that could arise from splicing intermediates, i.e. 3’ ends of annotated exons and introns.
?. [1.5.3 Determine RNA polymerase coverage genome-wide from read alignments.]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 1 | Western blot control: checking cell fractionation]
Separate samples by standard SDS-PAGE (loading 10 µl of each boiled sample per lane is usually sufficient).
?. [Box 1 | Western blot control: checking cell fractionation]
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).
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
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.
?. [Box 2 | Fragmentation test]
Prepare all RNA samples by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.
?. [Box 2 | Fragmentation test]
Denature the RNA sample and RNA ladder for 2 min at 80 °C. Place the samples on ice.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
Load gel with the RNA ladder and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.
?. [Box 2 | Fragmentation test]
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).
?. [Box 2 | Fragmentation test]
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).
?. [Box 3 | Rapid gel extraction]
Use a 21G needle to pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube.
?. [Box 3 | Rapid gel extraction]
Place the pierced 0.5-ml tube into a 1.5-ml RNase-free microcentrifuge tube.
?. [Box 3 | Rapid gel extraction]
Put the gel slice (from fragmentation, RT, or PCR) into the pierced 0.5-ml tube.
?. [Box 3 | Rapid gel extraction]
Centrifuge the nestled tubes, containing the gel slice, at 20,000g for 4 min at room temperature.
?. [Box 3 | Rapid gel extraction]
Add 200 µl of RNase-free H2O to the chopped-up gel and mix.
?. [Box 3 | Rapid gel extraction]
Incubate the sample mixture for 10 min at 70 °C in a Thermomixer.
?. [Box 3 | Rapid gel extraction]
Vortex the mixture for 30 s at medium intensity.
?. [Box 3 | Rapid gel extraction]
Cut the tip off of a 1,000-µl pipette tip and transfer the gel mixture into a microcentrifuge tube filter.
?. [Box 3 | Rapid gel extraction]
Centrifuge the mixture at 20,000g for 3 min at room temperature.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
For each PCR sample, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.
?. [Box 4 | PCR amplification test]
Add 1 µl of circularized cDNA and mix it well.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
Add 3.4 µl of 6× DNA loading dye to each tube and mix well.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
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.
?. [Box 4 | PCR amplification test]
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).
?. [Box 4 | PCR amplification test]
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).
?. [1.4 Constructing DNA sequencing library from isolated nascent RNA (Figure 1b)]
?. [Box 2 | Fragmentation test]
?. [Box 3 | Rapid gel extraction]
?. [Box 4 | PCR amplification test]
?. [1.4.4 Reverse transcription (Figure 3)]
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.
?. [1.4.1 Linker ligation (Figure 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.
?. [1.4.7 cDNA Circularization (Figure 4)]
Prepare circularization mix (see Materials section 1.4.7, Circularization of cDNA, step 2) and store it on ice.
?. [1.5 Bioinformatics analysis (Figure 5)]
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).
?. [Box 2 | Fragmentation test]
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.
?. [Box 3 | Rapid gel extraction]
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).
?. [Box 4 | PCR amplification test]
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.
?. [1.4.1 Linker ligation (Figure 2)]
Spray down all work surfaces, pipettes, etc. with RNase away
?. [1.3 Cellular Fractionation (Figure 1a)]
?. [1.4.1 Linker ligation (Figure 2)]
?. [1.4.4 Reverse transcription (Figure 3)]
?. [1.4.7 cDNA Circularization (Figure 4)]
?. [1.5 Bioinformatics analysis (Figure 5)]