TITLE: Transient transcriptome sequencing: experimental protocol to monitor genome-wide RNA synthesis including enhancer transcription
AUTHORS: Saskia Gressel, Katja Lidschreiber, Patrick Cramer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Transcriptome analysis by RNA sequencing (RNA-seq) measures the steady-state abundance of cellular RNA, which is a result of the interplay of RNA synthesis and RNA degradation. In order to measure RNA synthesis, RNA can be labeled with 4-thiouridine (4sU) in cells, purified and sequenced (4sU-seq). Although 4sU-seq has a higher sensitivity than RNA-seq, it is not sensitive enough to reliably detect short-lived (transient) RNAs such as enhancer, antisense, and promoter-associated RNAs synthesized from large genomes such as the human genome. This is because when the 4sU labeling time is less than 30 min only a short 3’-region of transcripts is labeled, and a long pre-existing unlabeled 5’ region leads to a 5’-bias in the sequencing data. Transient transcriptome sequencing (TT-seq) overcomes this limitation by combining a short 4sU labeling pulse with an RNA fragmentation step. The labeled, newly synthesized RNA fragments are purified and sequenced, resulting in a very low fraction of contaminating non-labeled RNA. TT-seq is easy to use and includes RNA spike-in controls for global normalization between datasets from different samples. TT-seq enables studies of the kinetics of RNA metabolism, and mechanistic studies of transcription regulation. Also, TT-seq is ideally suited to monitor rapid changes in gene activity as well as the dynamics of enhancer landscapes during transcription responses.</div><style>
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[STEPS]
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Perform first strand cDNA synthesis using the Transcriptor First Strand cDNA Synthesis Kit with Anchored-oligo (dT) primers. Follow manufacturer’s instructions, use  ERCC RNA Spike-in Mix.
1 µl
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Amplify specific spike-ins from  of 1:4 diluted cDNA using KOD Hot Start DNA Polymerase following manufacturer’s instructions (30 cycles).Primer sequences to amplify the cDNA template for IVT reaction of RNA spike-ins.  ABC1Primer name (TT-seq)Sequence 5'>3'Annealing temperature (°C)2Spike-in 1 forwardTAATACGACTCACTATAGGGTGCTTTAACAAGAGGAAATTGTGT533Spike-in 1 reverseCCATCTTGTTTATAAAATCCTAATTACTC534Spike-in 2 forwardTAATACGACTCACTATAGGGGGCACAAGTTGCTGAAGTTGC555Spike-in 2 reverseTCTGCTGTAATCTCAGCTCC556Spike-in 3 forwardTAATACGACTCACTATAGGGTTTCGACGTTTTGAAGGAGGG537Spike-in 3 reverseGTACCCGGGAAAATCCTAGTTC538Spike-in 4 forwardTAATACGACTCACTATAGGGACTGTCCTTTCATCCATAAGCGG559Spike-in 4 reverseCGCACGCCGAATGATGAAACG5510Spike-in 5 forwardTAATACGACTCACTATAGGGGATGTCCTTGGACGGGGT5511Spike-in 5 reverseGCTTTCGGAGCAAATCGCG5512Spike-in 6 forwardTAATACGACTCACTATAGGGCCAGATTACTTCCATTTCCGCC5513Spike-in 6 reverseGGGTAAAACGCAAGCACCG55
1 µl
ABC1Primer name (TT-seq)Sequence 5'>3'Annealing temperature (°C)2Spike-in 1 forwardTAATACGACTCACTATAGGGTGCTTTAACAAGAGGAAATTGTGT533Spike-in 1 reverseCCATCTTGTTTATAAAATCCTAATTACTC534Spike-in 2 forwardTAATACGACTCACTATAGGGGGCACAAGTTGCTGAAGTTGC555Spike-in 2 reverseTCTGCTGTAATCTCAGCTCC556Spike-in 3 forwardTAATACGACTCACTATAGGGTTTCGACGTTTTGAAGGAGGG537Spike-in 3 reverseGTACCCGGGAAAATCCTAGTTC538Spike-in 4 forwardTAATACGACTCACTATAGGGACTGTCCTTTCATCCATAAGCGG559Spike-in 4 reverseCGCACGCCGAATGATGAAACG5510Spike-in 5 forwardTAATACGACTCACTATAGGGGATGTCCTTGGACGGGGT5511Spike-in 5 reverseGCTTTCGGAGCAAATCGCG5512Spike-in 6 forwardTAATACGACTCACTATAGGGCCAGATTACTTCCATTTCCGCC5513Spike-in 6 reverseGGGTAAAACGCAAGCACCG55
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Six synthetic RNA spike-in controls (three unlabeled and three 4sU labeled spike-ins) were established for validation of labeled RNA enrichment, global normalization and for estimating possible cross-contamination from unlabeled RNA fragments. The spike-ins are derived from selected RNAs of the ERCC RNA Spike-in Mix (Schwalb et al. 2016). We chose six spike-ins of about 1,000 nt length, a GC content between 40 to 60 % and similar uridine content. The IVT simulates the in vivo labeling of the cells, assuming that one out of ten uridine bases is substituted with 4sU. If polyadenylated RNA spike-ins are required, add a poly(A) tailing step after PCR amplification, or perform the IVT directly from custom-made DNA sequences containing poly(A) tails.Selected RNA spike-ins derived from the ERCC RNA Spike-in Mix.  ABCDEF1ERCC IDLength (nt)%UTP%4sUTP%GCName (TT-seq)200043 985901034Spike-in 1300170949100035Spike-in 24001361,014901043Spike-in 35001451,015100046Spike-in 46000921,079901052Spike-in 57000021,037100053Spike-in 6
ABCDEF1ERCC IDLength (nt)%UTP%4sUTP%GCName (TT-seq)200043 985901034Spike-in 1300170949100035Spike-in 24001361,014901043Spike-in 35001451,015100046Spike-in 46000921,079901052Spike-in 57000021,037100053Spike-in 6
Steps 8-12 can be omitted by ordering custom-made DNA sequences.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Purify PCR products with QIAquick PCR purification columns following manufacturer’s instructions. Elute in .
30 µl
?. [Cell Treatment and 4sU Labeling]
The required number of cells depends on the cell line under study. The exact cell count is required to calculate the corresponding amount of RNA spike-ins.
Depending on cell type specific parameters (doubling time, transcriptional activity, and amount of cellular RNA), between 300 to 600 µg total RNA are needed to isolate sufficient amounts of labeled RNA (> 50 ng). To establish a 4sU-labeling experiment, determine first how many cells are needed to yield 300 (or 600 µg) of total RNA and then, how much labeled RNA can be isolate from 300 µg (or 600 µg) input RNA. Based on these results, calculate the required number of cells. Typically, for 5 min of 4sU labeling the required number of cells is 1.5 to 5 x 107.  At the time of labeling cells should still be in their exponential growth phase to ensure high transcriptional activity. Therefore, we label cells at around 70 % confluence. 
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?. [Cell Treatment and 4sU Labeling]
Labeling of Adherent Cells
?. [Cell Treatment and 4sU Labeling]
Optional: add treatment prior to 4sU labeling.
Labeling of control and treated cells: when comparing TT-seq of control and manipulated cells it is critical that cell densities and labeling times are kept consistent for comparable 4sU uptake and incorporation.
?. [Cell Treatment and 4sU Labeling]
24-48 h before the experiment, plate the required number of cells in 15 cm dishes ( culture medium). Seed 1-2 additional plates for cell counting.
20 ml
?. [Cell Treatment and 4sU Labeling]
before labeling thaw 4sU stock solution on ice and count cells.
?. [Cell Treatment and 4sU Labeling]
For 4sU labeling, incubate cells with a final concentration of  4sU at ,  CO2 for exactly .
500
5
4sU labeling of adherent cells: just before labeling, pipet the required amount of 4sU for each plate into a sterile 50 mL tube. Remove half of the culture medium from each plate (it is important to leave some volume behind to not stress the cells) and add to the 50 mL 4sU-tube, mix and immediately pour back. Do not handle more than three plates in parallel.
4sU concentration and labeling time can be adjusted to yield more labeled RNA. However, it is important to balance labeling concentration and time to avoid possible inhibition of rRNA synthesis (Burger et al. 2013) and cell toxicity.
?. [Cell Treatment and 4sU Labeling]
After  quickly remove culture medium and add  of TRIzol lysis reagent.
4 ml
Please refer to safety data sheet for hazardous identification of TRIzol lysis reagent. Always wear gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical hood. Avoid breathing vapor.
?. [Cell Treatment and 4sU Labeling]
"Wash" cells off the plate by pipetting up and down. If cells are very adherent, use a TRIzol compatible cell scraper.
?. [Cell Treatment and 4sU Labeling]
Transfer cell lysate to 15 mL polypropylene (PP) centrifuge tube and incubate up to .
Use centrifuge tubes for high speed (≥13,000 g) centrifugation.
If several plates are labeled simultaneously, two lysates from the same condition can be pooled into one PP tube (minimizes the number of tubes).
?. [Cell Treatment and 4sU Labeling]
Optional: store lysates at .
?. [Cell Treatment and 4sU Labeling]
Labeling of Suspension Cells
?. [Cell Treatment and 4sU Labeling]
Dilute cells 48 h before the labeling experiment and exchange growth medium one day before labeling.
?. [Cell Treatment and 4sU Labeling]
before labeling thaw 4sU stock solution on ice and count cells.
?. [Cell Treatment and 4sU Labeling]
Add  of 4sU stock solution to cells in  of growth medium to a final concentration of , and incubate at ,  CO2.
500 µl
50 ml
500
5
?. [Cell Treatment and 4sU Labeling]
Exactly after  of labeling, centrifuge at  and 500-1,500 x g for . Discard supernatant by decanting.
?. [Cell Treatment and 4sU Labeling]
Add  of TRIzol lysis reagent to cell pellet. Vortex until no cells are visible ().
5 ml
?. [Cell Treatment and 4sU Labeling]
Incubate for . Transfer to 15 mL PP centrifuge tubes.
Use centrifuge tubes for high speed (≥13,000 x g) centrifugation.
?. [Cell Treatment and 4sU Labeling]
Optional: store lysates at .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
If lysates were frozen, thaw quickly at  (water bath), then cool on ice for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Thaw RNA Spike-in mix at  for , cool on ice for  and mix gently by pipetting.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Add  spike-in mix per 106 cells to TRIzol cell lysate.
2
RNA spike-in addition to cell lysate: the amount of RNA spike-ins depends on the complexity and concentration of the final sample, but also on how deep the library is sequenced. In general, a balance between detecting the spike-ins and not having too many sequencing reads for spike-ins is recommended.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Vortex gently and incubate for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Add  of chloroform per  of TRIzol lysis reagent.
0 ml
1 ml
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Shake vigorously for  and incubate for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Centrifuge at  and  for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Carefully transfer upper, aqueous phase into new 15 mL centrifuge tube.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Add an equal volume of isopropanol to precipitate RNA, vortex gently.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Incubate for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Centrifuged at  and  for .
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Wash RNA pellet twice, first using an equal volume, then  of  ethanol. Spin in-between at  and  for .
1 ml
75
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Remove supernatant with 1 mL pipette. Spin down briefly and use 200 μL pipette to remove remaining ethanol. Eventually, spin down again and use 10 μL pipette.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Dissolve pellet in 200-1,000 µL H2O (aim for a final concentration of ≥ 750 ng/μL).
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Transfer RNA to 1.5 mL tube.
Optional: pool samples from the same condition into one tube.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Determine concentration and purity by NanoDrop spectrophotometer; 260/280 value should be >2.
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Save  of  total RNA for quality control on Agilent Bioanalyzer RNA 6000 Nano Chip (Step 45).
1 µl
?. [RNA Spike-in Pool Addition and Total RNA Extraction]
Optional: store at .
?. [ Mild RNA Fragmentation (based on Schwalb et al. 2016)]
Cool Bioruptor Plus to  before use. Transfer  of total RNA to 1.5 mL Bioruptor Plus TPX microtubes, adjust to .
300 µg
750
If processing   RNA: use two 1.5 mL Bioruptor Plus TPX microtubes (  each).
RNA fragmentation using Covaris S220: transfer two-times   of total RNA (each in a final volume of  ) to 130 µL microTUBE AFA Fiber Snap-Cap tubes. Use the following settings: water level 12, temperature , peak incident power 100 W, duty factor 1 %, cycles per burst 200, treatment time 10-60 sec.
?. [ Mild RNA Fragmentation (based on Schwalb et al. 2016)]
Fill empty positions in the tube holder with tubes containing  water.
400 µl
?. [ Mild RNA Fragmentation (based on Schwalb et al. 2016)]
Settings: 1 cycle, 30 sec ON, 30 sec OFF at HIGH power.
?. [ Mild RNA Fragmentation (based on Schwalb et al. 2016)]
Transfer (optional: pool) samples to 2 mL PP microtubes. Continue immediately with the biotinylation reaction.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Denature total fragmented RNA at  for , then place on ice for .
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Save  of total fragmented RNA for DNase I digest (Step 75).
5 µl
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Split remaining total fragmented RNA in two 2 mL PP microtubes (/reaction) and process in parallel in Steps 49-53.
150 µg
If processing   RNA: split total fragmented RNA in four 2 mL PP microtubes ( /reaction) and process in parallel (Steps 49-53).
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Add water up to .
700 µl
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Prepare biotin master mix in the stated order:  of 10x biotinylation buffer,  of EZ-link HPDP-Biotin stock solution.
100 µl
200 µl
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Add   biotin master mix to each sample and mix immediately.
300 µl
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Incubate in the dark with rotation for .
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Shortly before use, prepare 15 mL MaXtract High Density tubes following the manufacturer's instruction.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Transfer biotin-RNA mix to MaXtract High Density tubes, pool reactions from the same condition.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Add an equal volume of chloroform. Mix vigorously for  and incubate for .
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Centrifuge at  for .
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Transfer upper phase into 15 mL PP centrifuge tube.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
For RNA precipitation, add 1/10 volume of  NaCl and an equal volume of isopropanol. Vortex gently.
5
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Centrifuge at  and  for . Remove supernatant.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Wash RNA pellet twice, first using an equal volume, then  of  ethanol. Spin in-between at  and  for .
1 ml
75
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Remove supernatant with 1 mL pipette. Spin down briefly and use 200 μL pipette to remove remaining ethanol. Optionally, spin down again and use 10 μL pipette.
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Resuspend RNA in  H2O per  biotinylation reaction: chill on ice , then heat at  for , put on ice and carefully resuspend by pipetting. Transfer to 2 mL LoBind tubes.
100 µl
300 µg
?. [Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)]
Continue immediately with 4sU pull-down.
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Use one µMACS column per  () RNA.
300 µg
200 µl
If processing   RNA: use two µMAC columns with   ( ) RNA each.
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Heat µMACS wash buffer (/µMAC column) to .
3 ml
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Add  of µMACS streptavidin beads to  RNA.
100 µl
200 µl
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Incubate at  with rotation for .
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
In the meantime, equilibrate µMACS columns with  of nucleic acids equilibration buffer (equilibrate at RT  before use).
100 µl
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Transfer RNA-bead-mix to the µMACS column, collect the flow-through in LoBind tubes and reapply to the µMACS column. Discard flow-through.
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Wash 3-times with  of µMACS wash buffer (). Discard flow-through.
900 µl
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Wash 3-times with  of µMACS wash buffer (). Discard flow-through.
900 µl
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Place 1.5 ml LoBind tubes underneath the columns.
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
Elute labeled RNA in  of  DTT.
100 µl
100
?. [Pull-down of 4sU Labeled RNA Using Streptavidin-Beads]
After  elute in additional  of  DTT.
100 µl
100
?. [Final RNA Clean-up and DNase Digest]
Transfer samples onto miRNeasy MinElute spin column in a 2 mL collection tube.
If processing labeled RNA isolated from   total fragmented RNA: pool samples from the same condition onto one RNeasy MinElute spin column by repeating Step 78.
?. [Final RNA Clean-up and DNase Digest]
Centrifuge at  for . Reload flow-through.
?. [Final RNA Clean-up and DNase Digest]
Wash with  of RWT buffer by centrifugation at  for .
350 µl
?. [Final RNA Clean-up and DNase Digest]
Prepare DNase mix:  RDD buffer and  RNase-free DNase I, mix by pipetting or inverting.
70 µl
10 µl
?. [Final RNA Clean-up and DNase Digest]
Add  DNase master mix to each column and incubate for .
80 µl
?. [Final RNA Clean-up and DNase Digest]
Add  of RWT buffer.
500 µl
?. [Final RNA Clean-up and DNase Digest]
Centrifuge at  for . Reload flow-through.
?. [Final RNA Clean-up and DNase Digest]
Centrifuge at  for . Discard flow-through.
?. [Final RNA Clean-up and DNase Digest]
Add  of RPE buffer.
500 µl
?. [Final RNA Clean-up and DNase Digest]
Centrifuge at  for .
?. [Final RNA Clean-up and DNase Digest]
Place column into a new 2 mL collection tube.
?. [Final RNA Clean-up and DNase Digest]
Wash with  of  ethanol. Centrifuge at  for . Place column into new 2 mL collection tube.
500 µl
80
?. [Final RNA Clean-up and DNase Digest]
To dry the membrane, open the lid and centrifuge at full speed for .
?. [Final RNA Clean-up and DNase Digest]
Place column into new 1.5 mL LoBind tube.
?. [Final RNA Clean-up and DNase Digest]
Elute with  of nuclease free H2O (not DEPC treated) by centrifugation at full speed for . Reload flow-through for a second elution. If a low labeled RNA yield is expected: use  of H2O and incubate  before centrifugation.
15 µl
11 µl
?. [Final RNA Clean-up and DNase Digest]
Transfer RNA to a new 1.5 mL LoBind tube.
?. [Final RNA Clean-up and DNase Digest]
Measure RNA concentration with Qubit RNA HS Kit and analyze RNA integrity on Agilent Bioanalyzer RNA 6000 Pico Chip.
?. [Expected Result]
Highly recommended: validation of labeled RNA enrichment by reverse transcription PCR (RT-qPCR) of RNA spike-ins.Primer sequences for RT-qPCR of RNA spike-ins to control enrichment of labeled RNA.  ABCD1Primer name (TT-seq)Sequence 5'>3'Annealing temperatureAmplicon size (bp)2Spike-in 1 forwardACAATTCCAAATAGCGACCACATCA591503Spike-in 1 reverseTACCTCAACCCTTCCAGTGTCTAAG584Spike-in 2 forwardAGACTGGCATTCCCGTGATA57975Spike-in 2 reverseGCTAAAACCCCTGCCTGCAA606Spike-in 3 forwardCCGAGTTCGCCTTACTGCTC60957Spike-in 3 reverseAATCGATCGGAATCACGCCG608Spike-in 4 forwardCATAAGCGGAGAAAGAGGGAATGAC591039Spike-in 4 reverseGCTAAATAGAGAGCATCCACACCTC5810Spike-in 5 forwardCGTTAATGCAGAGGCTAAGGACAAT5910311Spike-in 5 reverseGATCGTTACAAACCCACTACGTGTC5912Spike-in 6 forwardGTCCTGATTTACTGGACTCGCAAC5811813Spike-in 6 reverseTCTGTATAAGGTGATCGCAGGTTGT59
Control of labeled RNA enrichment by RT-qPCR. Prepare cDNA from   total fragmented RNA and   labeled RNA, and perform RT-qPCR. Use primer pairs for RNA spike-ins to estimate labeled RNA enrichment, and (optional) for endogenous model genes of your choice to measure the intron enrichment.
ABCD1Primer name (TT-seq)Sequence 5'>3'Annealing temperatureAmplicon size (bp)2Spike-in 1 forwardACAATTCCAAATAGCGACCACATCA591503Spike-in 1 reverseTACCTCAACCCTTCCAGTGTCTAAG584Spike-in 2 forwardAGACTGGCATTCCCGTGATA57975Spike-in 2 reverseGCTAAAACCCCTGCCTGCAA606Spike-in 3 forwardCCGAGTTCGCCTTACTGCTC60957Spike-in 3 reverseAATCGATCGGAATCACGCCG608Spike-in 4 forwardCATAAGCGGAGAAAGAGGGAATGAC591039Spike-in 4 reverseGCTAAATAGAGAGCATCCACACCTC5810Spike-in 5 forwardCGTTAATGCAGAGGCTAAGGACAAT5910311Spike-in 5 reverseGATCGTTACAAACCCACTACGTGTC5912Spike-in 6 forwardGTCCTGATTTACTGGACTCGCAAC5811813Spike-in 6 reverseTCTGTATAAGGTGATCGCAGGTTGT59
For total fragmented RNA, the Ct values of labeled and unlabeled RNA spike-ins are very similar. Labeled RNA shows a Ct value difference of 6-10 for labeled and unlabeled RNA spike-ins. For the selected model gene, intron levels should be enriched in labeled RNA compared to total fragmented RNA.
?. [Library Preparation]
We recommend using  (at least ) labeled RNA and  total fragmented RNA as library input.
100
50
100
We recommend using the Ovation Universal RNA-Seq System (NuGEN). The option of using only random primers for cDNA synthesis omits the 3' bias generated by poly-(dT) primers. Also, the NuGEN kit depletes ribosomal RNA by sequence-specific probes (AnyDeplete technology, formerly known as InDA-C).
Total fragmented RNA samples are needed to estimate RNA half-lives and correct the cross-contamination using RNA spike-ins. If the cross-contamination rate is similar across your libraries (which can be estimated from the RNA spike-ins), labeled RNA might be sufficient.
?. [Library Preparation]
For ‘First Strand Primer Pre-mix Preparation’, use only Random primer mix. Substitute poly-d(T) primers with  H2O.
0 µl
?. [Library Preparation]
Follow the User guide’s instruction from ‘First Strand Synthesis Using DNase-treated RNA’ to ‘Second Strand cDNA Synthesis’.
?. [Library Preparation]
For ‘cDNA fragmentation’ (200-400 bp) using a Bioruptor Plus device: Aliquot  cDNA in Qubit assay tubes. Fill up empty positions in the tube holder with tubes containing  water. Do 15 cycles, 30 sec ON, 30 sec OFF at LOW power. Spin down samples and add another 10 cycles. If the Bioruptor is not connected to an automated cooling system. Cool the water bath with ice in-between the 15 and 10 cycles.
100 µl
100 µl
cDNA fragmentation using Covaris S220 (200-400 bp): transfer cDNA to 130 µL microTUBE AFA Fiber Snap-Cap tube. Use the following settings: water level 12, temperature , peak incident power 145 W, duty factor 10 %, cycles per burst 200, treatment time 50-180 sec.
?. [Library Preparation]
Follow the User guide’s instruction from ‘cDNA Concentration After Fragmentation’ to ‘Adaptor Cleavage’.
?. [Library Preparation]
Before ‘PCR amplification’: To avoid over-amplification determine the precise number of PCR cycles by the KAPA HIFI Library Amp Real Time kit following manufacturer’s instructions. As input, use  (~10 %) of the library,  of 2x Kapa HiFi Hot Start Master Mix and  Amplification Primer Mix P2 (NuGEN).
4 µl
5 µl
0 µl
?. [Library Preparation]
For ‘PCR amplification’ of the remaining  of library use the determined number of PCR cycles and follow the User guide’s instructions for amplification as described in the Appendix ‘Using qPCR to determine the Number of PCR Cycles’.
45 µl
?. [Library Preparation]
Proceed from ‘Bead Purification of the Amplified Material’ to ‘Quantitative and Qualitative Assessment of the Library’.
?. [Library Preparation]
Assess the quality of the library on Bioanalyzer DNA 1000 Chip and send to sequencing service providers for high throughput sequencing.
?. [Paired-end Illumina Sequencing]
Universal RNA-Seq with NuQuantThe read length for human transcriptomes should be between 35 to 150 bp depending on the desired application. For alternative splicing detection, longer reads are recommended, i.e. 150 bp paired-end sequencing. Recommended coverage: in general, 25 to 50 million reads per sample are sufficient for an initial analysis of highly expressed transcripts. 100 million reads is the required sequencing depth for annotation of enhancer RNAs or antisense RNAs in TT-seq (labeled RNA) samples (see Deveson et al. 2017 for review). If the focus of a study is on newly synthesized RNA fragments, total fragmented RNA-seq libraries can be sequenced with a lower coverage, i.e. 30 million reads. If a study aims to study RNA stability, TT-seq and total fragmented RNA-seq libraries should be sequenced at similar coverage.
?. [Computational analysis]
The bioinformatics workflow used to analyze TT-seq data is presented in Villamil et al. 2019. The pre-print describe pre-processing steps, including a reliable and robust normalization strategy, and several downstream analysis tools that enable the user to quantify RNA synthesis, splicing and degradation activities.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Control size and purity of PCR products by gel electrophoresis.
Optional cDNA purification: if PCR products show contamination with smaller fragments, isolate the PCR product of the desired size by gel extraction (e.g. by QIAquick Gel Extraction Kit).
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Verify sequence of PCR products by Sanger sequencing.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Use  PCR product (or, custom-made DNA) as input for in vitro transcription (IVT) using the MEGAscript T7 kit. Follow manufacturer’s instructions, except for IVT of labeled RNA spike-ins (referred to as 1, 3, 5) substitute  of UTP with 4-Thio-UTP.
0 µg
10
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Purify RNA spike-ins with RNase-free AMPure XP beads following manufacturer’s instructions.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Use the Qubit HS RNA Kit and Agilent RNA 6000 Nano Kit for quantification and quality assessment.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
For the final RNA spike-in pool containing equimolar amounts of all six RNA spike-ins, add each RNA spike-in to a final concentration of .
1
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Assess the actual concentration of your RNA spike-in pool by Qubit HS RNA Kit and use this concentration as your working concentration.
?. [Spike-in Pool Preparation (based on Schwalb et al. 2016)]
Store RNA spike-in pool in small, one-time use aliquots at  (see Step 25 for the required amount of spike-ins per sample).
?. [ Mild RNA Fragmentation (based on Schwalb et al. 2016)]
After fragmentation, use  of total fragmented RNA for quality control on Agilent Bioanalyzer RNA 6000 Nano Chip and compare to  of total RNA. For expected results see Step 94.
1 µl
1 µl
?. [Expected Result]
Examples of RNA integrity profiles of total, total fragmented and labeled RNA isolated from human cell lines.
Total fragmented RNA should be in a range between 0.2 and 15 kbp. Fragmentation efficiency might differ for RNA from another organisms. Thus, it is recommended to perform initial trials with your cell lines. Be aware that after sonication it is expected that the RIN value drops since rRNA is also fragmented.
?. [Final RNA Clean-up and DNase Digest]
Prepare labeled RNA (Step 74). Mix samples thoroughly by shaking for .  AB14sU labeled RNA200 µL2100 % ethanol300 µL
AB14sU labeled RNA200 µL2100 % ethanol300 µL
?. [Final RNA Clean-up and DNase Digest]
Prepare  total fragmented RNA (Step 47). Mix samples thoroughly by shaking for .  AB1Total fragmented RNA (1 µg)200 µL23 M NaAc pH 5.215 µL3100 % ethanol300 µL
1 µg
AB1Total fragmented RNA (1 µg)200 µL23 M NaAc pH 5.215 µL3100 % ethanol300 µL
From here on use low binding pipette tips. Process labeled RNA and   of total fragmented RNA in parallel.
?. [Buffers and Stock Solutions]
Prepare  4-thiouridine (4sU) stock solution. Dissolve 4sU in sterile RNase-free PBS. Store in small aliquots at .
50
Thaw 4sU only once, and use immediately. As 4sU is photoactivatable and crosslinks to proteins at 365 nm, avoid light sources that emit this wavelength. The incorporation of 4sU can be validated by dot blot (see e.g. Radle et al. 2013).
?. [Buffers and Stock Solutions]
Prepare 10x biotinylation buffer containing  Tris HCl pH 7.4 and  EDTA pH 8.0. Store in small aliquots at .
100
10
?. [Buffers and Stock Solutions]
Prepare EZ-link HPDP-biotin stock solution. EZ-link HPDP-biotin is pyridyldithiol-activated and allows thiol-specific biotinylation of labeled RNA. Dissolve in dimethylformamide (DMF) to , mix thoroughly and incubate at  until it is completely dissolved. Repeat mixing if needed. Store in small aliquots in 2 mL PP micro tubes at .
1
Please refer to safety data sheet for hazardous identification of DMF. Always wear gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical hood. Avoid breathing vapor. DMF should not get in contact with incompatible plastic materials. Eluted plastic might be carried along and causes substantial loss of labeled RNA.
Regarding the use of MTS-biotin (Duffy et al. 2015) in replacement of HPDP-biotin. We performed extensive control experiments to evaluate the described use of MTS-biotin in replacement of HPDP-biotin for our TT-seq protocol. For instance, we  used 4sU-free total cellular RNA spiked-in with 0.1 % of 4sU labeled spike-ins, to assess the specificity of both biotins. When using MTS-biotin we found high levels of cross-contamination in the 4sU-pulldown fraction with unlabeled cellular RNA. This is in agreement with Marzi and Nicassio 2018 who detected 10-fold higher background levels for MTS-biotin compared to HPDP-biotin. Recently an improved MTS-biotin protocol including optimized biotin chemistry (MTS-resin) was developed (Duffy et al. 2018). As we have not yet tested this improved biotinylation protocol, we recommend to carry out careful control experiments before using this alternative for TT-seq.
?. [Buffers and Stock Solutions]
Prepare µMACS wash buffer containing  Tris HCl pH 7.4,  EDTA pH 8.0,  NaCl,  (vol/vol) Tween20.
100
10
1
0
?. [Buffers and Stock Solutions]
Prepare   DL-dithiothreitol (DTT) in RNAse-free H2O. Store in small aliquots at . Thaw only once, and dilute to  before use.
1
100
?. [Buffers and Stock Solutions]
Prepare miRNeasy Micro Kit buffer RWT with isopropanol instead of ethanol (see manufacturer's instructions for “DNase digestion for samples containing