Protocol Citation: Saskia Gressel, Katja Lidschreiber, Patrick Cramer 2019. Transient transcriptome sequencing: experimental protocol to monitor genome-wide RNA synthesis including enhancer transcription. protocols.io https://dx.doi.org/10.17504/protocols.io.3vzgn76
Manuscript citation:
This manuscript will be published as a chapter in Methods in Molecular Biology.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Here, we describe an easy-to-use, detailed bench protocol for application of TT-seq, including in vitro transcribed RNA spike-in controls that enable normalization across samples, estimation of the amount of cross-contamination by non-labeled RNA, and a correction for antisense artifacts. This manuscript will be published as a chapter in Methods in Molecular Biology.
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.
Bioanalyzer DNA 1000 ChipAgilent TechnologiesCatalog #5067
All materials, consumables and chemicals must be sterile, RNase-free, molecular biology grade.
It is essential to use RNase-free equipment.
Buffers and Stock Solutions
Buffers and Stock Solutions
Prepare 50 mM4-thiouridine (4sU) stock solution. Dissolve 4sU in sterile RNase-free PBS. Store in small aliquots at -20 °C.
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 1 mg/mL, mix thoroughly and incubate at 37 °C until it is completely dissolved. Repeat mixing if needed. Store in small aliquots in 2 mL PP micro tubes at -20 °C.
Prepare 10x biotinylation buffer containing 100 mM Tris HCl pH 7.4 and 10 mM EDTA pH 8.0. Store in small aliquots at 4 °C.
Prepare µMACS wash buffer containing 100 mM Tris HCl pH 7.4, 10 mM EDTA pH 8.0, 1 M NaCl, 0.1 % (vol/vol) Tween20.
Prepare 1 MDL-dithiothreitol (DTT) in RNAse-free H2O. Store in small aliquots at -20 °C. Thaw only once, and dilute to 100 mM before use.
Prepare miRNeasy Micro Kit buffer RWT with isopropanol instead of ethanol (see manufacturer's instructions for “DNase digestion for samples containing <1 μg total RNA approximately”).
Spike-in Pool Preparation (based on Schwalb et al. 2016)
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.
ERCC ID
Length (nt)
%UTP
%4sUTP
%GC
Name (TT-seq)
00043
985
90
10
34
Spike-in 1
00170
949
100
0
35
Spike-in 2
00136
1,014
90
10
43
Spike-in 3
00145
1,015
100
0
46
Spike-in 4
00092
1,079
90
10
52
Spike-in 5
00002
1,037
100
0
53
Spike-in 6
Perform first strand cDNA synthesis using the Transcriptor First Strand cDNA Synthesis Kit with Anchored-oligo (dT) primers. Follow manufacturer’s instructions, use 1 µL ERCC RNA Spike-in Mix.
Amplify specific spike-ins from 1 µL 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.
Primer name (TT-seq)
Sequence 5'>3'
Annealing temperature (°C)
Spike-in 1 forward
TAATACGACTCACTATAGGGTGCTTTAACAAGAGGAAATTGTGT
53
Spike-in 1 reverse
CCATCTTGTTTATAAAATCCTAATTACTC
53
Spike-in 2 forward
TAATACGACTCACTATAGGGGGCACAAGTTGCTGAAGTTGC
55
Spike-in 2 reverse
TCTGCTGTAATCTCAGCTCC
55
Spike-in 3 forward
TAATACGACTCACTATAGGGTTTCGACGTTTTGAAGGAGGG
53
Spike-in 3 reverse
GTACCCGGGAAAATCCTAGTTC
53
Spike-in 4 forward
TAATACGACTCACTATAGGGACTGTCCTTTCATCCATAAGCGG
55
Spike-in 4 reverse
CGCACGCCGAATGATGAAACG
55
Spike-in 5 forward
TAATACGACTCACTATAGGGGATGTCCTTGGACGGGGT
55
Spike-in 5 reverse
GCTTTCGGAGCAAATCGCG
55
Spike-in 6 forward
TAATACGACTCACTATAGGGCCAGATTACTTCCATTTCCGCC
55
Spike-in 6 reverse
GGGTAAAACGCAAGCACCG
55
Purify PCR products with QIAquick PCR purification columns following manufacturer’s instructions. Elute in 30 µL.
Control size and purity of PCR products by gel electrophoresis.
Verify sequence of PCR products by Sanger sequencing.
Use 0.5 µg 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 10 % of UTP with 4-Thio-UTP.
Purify RNA spike-ins with RNase-free AMPure XP beads following manufacturer’s instructions.
Use the Qubit HS RNA Kit and Agilent RNA 6000 Nano Kit for quantification and quality assessment.
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 ng/µL.
Assess the actual concentration of your RNA spike-in pool by Qubit HS RNA Kit and use this concentration as your working concentration.
Store RNA spike-in pool in small, one-time use aliquots at -80 °C (see Step 25 for the required amount of spike-ins per sample).
Cell Treatment and 4sU Labeling
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.
Optional: add treatment prior to 4sU labeling.
Labeling of Adherent Cells
24-48 h before the experiment, plate the required number of cells in 15 cm dishes (20 mL culture medium). Seed 1-2 additional plates for cell counting.
00:30:00 before labeling thaw 4sU stock solution on ice and count cells.
For 4sU labeling, incubate cells with a final concentration of 500 µM 4sU at 37 °C, 5 % CO2 for exactly 00:05:00.
After 00:05:00 quickly remove culture medium and add 4.5 mL of TRIzol lysis reagent.
"Wash" cells off the plate by pipetting up and down. If cells are very adherent, use a TRIzol compatible cell scraper.
Transfer cell lysate to 15 mL polypropylene (PP) centrifuge tube and incubate up to 00:05:00.
Optional: store lysates at -80 °C.
Labeling of Suspension Cells
Dilute cells 48 h before the labeling experiment and exchange growth medium one day before labeling.
00:30:00 before labeling thaw 4sU stock solution on ice and count cells.
Add 500 µL of 4sU stock solution to cells in 50 mL of growth medium to a final concentration of 500 µM, and incubate at 37 °C, 5 % CO2.
Exactly after 00:05:00 of labeling, centrifuge at 37 °C and 500-1,500 x g for 00:02:00. Discard supernatant by decanting.
Add 5 mL of TRIzol lysis reagent to cell pellet. Vortex until no cells are visible (00:00:30).
Incubate for 00:05:00. Transfer to 15 mL PP centrifuge tubes.
Optional: store lysates at -80 °C.
RNA Spike-in Pool Addition and Total RNA Extraction
RNA Spike-in Pool Addition and Total RNA Extraction
If lysates were frozen, thaw quickly at 65 °C (water bath), then cool on ice for 00:05:00.
Thaw RNA Spike-in mix at 65 °C for 00:02:00, cool on ice for 00:01:00 and mix gently by pipetting.
Add 2.4 ng spike-in mix per 106 cells to TRIzol cell lysate.
Vortex gently and incubate for 00:05:00.
Add 0.2 mL of chloroform per 1 mL of TRIzol lysis reagent.
Shake vigorously for 00:00:15 and incubate for 00:03:00.
Centrifuge at 4 °C and 13000 x g for 00:15:00.
Carefully transfer upper, aqueous phase into new 15 mL centrifuge tube.
Add an equal volume of isopropanol to precipitate RNA, vortex gently.
Incubate for 00:10:00.
Centrifuged at 4 °C and 13000 x g for 00:10:00.
Wash RNA pellet twice, first using an equal volume, then 1 mL of 75 % ethanol. Spin in-between at 4 °C and 13000 x g for 00:10:00.
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.
Dissolve pellet in 200-1,000 µL H2O (aim for a final concentration of ≥ 750 ng/μL).
Transfer RNA to 1.5 mL tube.
Determine concentration and purity by NanoDrop spectrophotometer; 260/280 value should be >2.
Save 1 µL of total RNA for quality control on Agilent Bioanalyzer RNA 6000 Nano Chip (Step 45).
Optional: store at -80 °C.
Mild RNA Fragmentation (based on Schwalb et al. 2016)
Mild RNA Fragmentation (based on Schwalb et al. 2016)
Cool Bioruptor Plus to 4 °C before use. Transfer 300 µg of total RNA to 1.5 mL Bioruptor Plus TPX microtubes, adjust to 750 ng/µL.
Fill empty positions in the tube holder with tubes containing 400 µL water.
Settings: 1 cycle, 30 sec ON, 30 sec OFF at HIGH power.
Transfer (optional: pool) samples to 2 mL PP microtubes. Continue immediately with the biotinylation reaction.
After fragmentation, use 1 µL of total fragmented RNA for quality control on Agilent Bioanalyzer RNA 6000 Nano Chip and compare to 1 µL of total RNA. For expected results see Step 94.
Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)
Biotinylation of 4sU Labeled RNA (based on Dölken et al. 2008)
Denature total fragmented RNA at 65 °C for 00:10:00, then place on ice for 00:05:00.
Save 5 µLof total fragmented RNA for DNase I digest (Step 75).
Split remaining total fragmented RNA in two 2 mL PP microtubes (150 µg/reaction) and process in parallel in Steps 49-53.
Add water up to700 µL.
Prepare biotin master mix in the stated order: 100 µL of 10x biotinylation buffer, 200 µL of EZ-link HPDP-Biotin stock solution.
Add 300 µL biotin master mix to each sample and mix immediately.
Incubate in the dark with rotation for 02:00:00.
Shortly before use, prepare 15 mL MaXtract High Density tubes following the manufacturer's instruction.
Transfer biotin-RNA mix to MaXtract High Density tubes, pool reactions from the same condition.
Add an equal volume of chloroform. Mix vigorously for 00:00:15 and incubate for 00:03:00.
Centrifuge at 1500 x g for 00:05:00.
Transfer upper phase into 15 mL PP centrifuge tube.
For RNA precipitation, add 1/10 volume of 5 M NaCl and an equal volume of isopropanol. Vortex gently.
Centrifuge at 4 °C and 13000 x g for 00:30:00. Remove supernatant.
Wash RNA pellet twice, first using an equal volume, then 1 mL of 75 % ethanol. Spin in-between at 4 °C and 13000 x g for 00:10:00.
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.
Resuspend RNA in 100 µL H2O per 300 µg biotinylation reaction: chill on ice 00:10:00, then heat at 65 °C for 00:10:00, put on ice and carefully resuspend by pipetting. Transfer to 2 mL LoBind tubes.
Continue immediately with 4sU pull-down.
Pull-down of 4sU Labeled RNA Using Streptavidin-Beads
Pull-down of 4sU Labeled RNA Using Streptavidin-Beads
Use one µMACS column per 300 µg (200 µL) RNA.
Heat µMACS wash buffer (3 mL/µMAC column) to 65 °C.
Add 100 µL of µMACS streptavidin beads to 200 µL RNA.
Incubate at 4 °C with rotation for 00:15:00.
In the meantime, equilibrate µMACS columns with 100 µL of nucleic acids equilibration buffer (equilibrate at RT 00:10:00 before use).
Transfer RNA-bead-mix to the µMACS column, collect the flow-through in LoBind tubes and reapply to the µMACS column. Discard flow-through.
Wash 3-times with 900 µL of µMACS wash buffer (65 °C). Discard flow-through.
Wash 3-times with 900 µL of µMACS wash buffer (Room temperature). Discard flow-through.
Place 1.5 ml LoBind tubes underneath the columns.
Elute labeled RNA in 100 µL of 100 mM DTT.
After 00:05:00 elute in additional 100 µL of 100 mM DTT.
Final RNA Clean-up and DNase Digest
Final RNA Clean-up and DNase Digest
Prepare 1 µg total fragmented RNA (Step 47). Mix samples thoroughly by shaking for 00:00:15.
Total fragmented RNA (1 µg)
200 µL
3 M NaAc pH 5.2
15 µL
100 % ethanol
300 µL
Prepare labeled RNA (Step 74). Mix samples thoroughly by shaking for 00:00:15.
4sU labeled RNA
200 µL
100 % ethanol
300 µL
Transfer samples onto miRNeasy MinElute spin column in a 2 mL collection tube.
Centrifuge at 8000 x g for 00:00:15. Reload flow-through.
Wash with 350 µL of RWT buffer by centrifugation at 8000 x g for 00:00:30.
Prepare DNase mix: 70 µL RDD buffer and 10 µL RNase-free DNase I, mix by pipetting or inverting.
Add 80 µL DNase master mix to each column and incubate for 00:15:00.
Add 500 µL of RWT buffer.
Centrifuge at 8000 x g for 00:00:30. Reload flow-through.
Centrifuge at 8000 x g for 00:00:30. Discard flow-through.
Add 500 µL of RPE buffer.
Centrifuge at 8000 x g for 00:00:30.
Place column into a new 2 mL collection tube.
Wash with 500 µL of 80 % ethanol. Centrifuge at 8000 x g for 00:02:00. Place column into new 2 mL collection tube.
To dry the membrane, open the lid and centrifuge at full speed for 00:05:00.
Place column into new 1.5 mL LoBind tube.
Elute with 15 µL of nuclease free H2O (not DEPC treated) by centrifugation at full speed for 00:01:00. Reload flow-through for a second elution. If a low labeled RNA yield is expected: use 11 µL of H2O and incubate 00:01:00 before centrifugation.
Transfer RNA to a new 1.5 mL LoBind tube.
Measure RNA concentration with Qubit RNA HS Kit and analyze RNA integrity on Agilent Bioanalyzer RNA 6000 Pico Chip.
Expected Result
Expected Result
Examples of RNA integrity profiles of total, total fragmented and labeled RNA isolated from human cell lines.
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.
Primer name (TT-seq)
Sequence 5'>3'
Annealing temperature
Amplicon size (bp)
Spike-in 1 forward
ACAATTCCAAATAGCGACCACATCA
59
150
Spike-in 1 reverse
TACCTCAACCCTTCCAGTGTCTAAG
58
Spike-in 2 forward
AGACTGGCATTCCCGTGATA
57
97
Spike-in 2 reverse
GCTAAAACCCCTGCCTGCAA
60
Spike-in 3 forward
CCGAGTTCGCCTTACTGCTC
60
95
Spike-in 3 reverse
AATCGATCGGAATCACGCCG
60
Spike-in 4 forward
CATAAGCGGAGAAAGAGGGAATGAC
59
103
Spike-in 4 reverse
GCTAAATAGAGAGCATCCACACCTC
58
Spike-in 5 forward
CGTTAATGCAGAGGCTAAGGACAAT
59
103
Spike-in 5 reverse
GATCGTTACAAACCCACTACGTGTC
59
Spike-in 6 forward
GTCCTGATTTACTGGACTCGCAAC
58
118
Spike-in 6 reverse
TCTGTATAAGGTGATCGCAGGTTGT
59
Library Preparation
Library Preparation
We recommend using 100 ng (at least 50 ng) labeled RNA and 100 ng total fragmented RNA as library input.
For ‘First Strand Primer Pre-mix Preparation’, use only Random primer mix. Substitute poly-d(T) primers with 0.4 µL H2O.
Follow the User guide’s instruction from ‘First Strand Synthesis Using DNase-treated RNA’ to ‘Second Strand cDNA Synthesis’.
For ‘cDNA fragmentation’ (200-400 bp) using a Bioruptor Plus device: Aliquot 100 µL cDNA in Qubit assay tubes. Fill up empty positions in the tube holder with tubes containing 100 µL 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.
Follow the User guide’s instruction from ‘cDNA Concentration After Fragmentation’ to ‘Adaptor Cleavage’.
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 4.2 µL (~10 %) of the library, 5 µL of 2x Kapa HiFi Hot Start Master Mix and 0.8 µL Amplification Primer Mix P2 (NuGEN).
For ‘PCR amplification’ of the remaining 45 µL 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’.
Proceed from ‘Bead Purification of the Amplified Material’ to ‘Quantitative and Qualitative Assessment of the Library’.
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
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
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.