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Transcriptome Dynamics via 4sU-Seq

Total RNA was extracted with the miRNeasy kit’s procedure (Qiagen), and sample quality was tested on a 2100 Bioanalyzer (Agilent). For the high-resolution (signature) analysis, we extracted RNA starting at 0h and until 3h after LPS stimulation in 15 minutes intervals. We generated replicated samples for the 0, 30, 45, 60 and 75 minutes samples. For the lower resolution analysis (4sU-Seq), we extracted RNA starting 0h and until 6h after LPS stimulation in 60 minutes intervals.
We used 20 μg total RNA for the biotinylation reaction. 4sU-labeled RNA was biotinylated using EZ-Link Biotin-HPDP (Pierce), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/mL, and stored at -80°C. Biotinylation was done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP for 2h at room temperature. Unbound Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1) extraction using MaXtract (high density) tubes (Qiagen). RNA was precipitated at 20,000g for 20 min with a 1:10 volume of 5M NaCl and an equal volume of isopropanol. The pellet was washed with an equal volume of 75% ethanol and precipitated again at 20,000g for 10 min. The pellet was re-suspended in 100 μL RNAse-free water. Biotinylated RNA was captured using Dynabeads MyOne™ Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 100 μL Dynabeads with rotation for 15 min at room temperature. Beads were magnetically fixed and washed with 1x Dynabeads washing buffer. Flow-through was collected for unlabeled preexisting RNA recovery. RNA-4sU was eluted with 100 μL of freshly prepared 100 mM dithiothreitol (DTT). RNA was recovered from eluates and washing fractions with RNeasy MinElute Spin columns (Qiagen).

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Rabani M., Levin J.Z., Fan L., Adiconis X., Raychowdhury R., Garber M., Gnirke A., Nusbaum C., Hacohen N., Friedman N., Amit I, & Regev A. (2011). Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nature biotechnology, 29(5), 436-442.
Publication 2011
Biotin Biotinylation Buffer Chloroform Dimethylformamide Dithiothreitol Edta Ethanol Hpdp biotin Isopropanol Nacl Rnase Streptavidin Tris

Corresponding Organization :

Other organizations : Broad Institute, Hebrew University of Jerusalem, Howard Hughes Medical Institute, Massachusetts Institute of Technology

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Protocol cited in 23 other protocols

1

Purification and Biotinylation of 4sU-Labeled RNA

Newly synthesised RNAs were purified as previously described in detail (Rädle et al., 2013 (link); Schwalb et al., 2016 (link); Rabani et al., 2011 (link)). Briefly, 4sU-labelled total RNA of spike-in cells (D. melanogaster) was added to 250 μg of labelled total RNA from mouse ESCs in a ratio 1:10 prior to newly synthesised RNA purification. The RNA was precipitated and resuspended in 130 μl RNase-free water (Sigma-Aldrich, Cat# 95284) and sonicated on a E220 Focused-ultrasonicator (Covaris) using the following settings: 1% duty factor, 100 W, 200 cycles per burst, 80 s, to obtain fragment size range from 10 kb to 200 bp. For purification, the fragmented total RNA was incubated for 10 min at 60°C and immediately chilled on ice for 2 min to open secondary RNA structures. Biotinylation was performed in labelling buffer (10 mM HEPES–KOH pH 7.5 and 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP (ThermoFisher Scientific, Cat# 21341) for 3 h at room temperature at 24°C in the dark and with gentle agitation. Unbound Biotin-HPDP was removed by adding an equal volume of chloroform/isoamyl alcohol (24:1) at 16000 g for 5 min at 4°C. RNA was precipitated at 20,000 g for 20 min with a 1:10 volume of 5 M NaCl and an equal volume of 100% isopropanol. The pellet was washed with an equal volume of 75% ethanol and precipitated again at 20,000 g for 10 min. The pellet was resuspended in 100 μL RNase-free water. Biotinylation and purification of 4sU-labelled RNAs was performed as described (Dölken et al., 2008 (link); Wachutka et al., 2019 (link)). Biotinylated RNA was captured using 100 μl of streptavidin-coated μMACS magnetic beads (Miltenyi Biotec, Cat# 130-074-101) for 90 min at 24°C under gentle agitation. The μMACS columns (Miltenyi Biotec, Cat# 130-074-101) were placed on a MACS MultiStand (Miltenyi Biotec) and equilibrated with washing buffer (100 mM Tris–HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20) twice on the columns before adding the samples. The columns were then washed once with 600 μl, 700 μl, 800 μl, 900 μl and 1 ml with washing buffer. Flow-through was collected for recovery of unlabeled preexisting RNA. RNA-4sU was eluted with two washes of 100 μL of freshly prepared 100 mM dithiothreitol (DTT). Reverse Transcription (RT) was performed with 1 μg total RNA and using 0.2 μg random hexamer primers (ThermoFisher Scientific, Cat# SO142) and 200 U SuperScript® IV Reverse Transcriptase (ThermoFisher Scientific, Cat# 18090050) following manufacturer’s instructions.
Forouzanfar F., Plassard D., Furst A., Moreno D., Oliveira K.A., Reina-San-Martin B., Tora L., Molina N, & Mendoza M. (2024). Gene-specific RNA homeostasis revealed by perturbation of coactivator complexes. bioRxiv.
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2

Nascent RNA-seq Using 4sU Labeling

Nascent RNA-seq using 4sU was performed as described previously (Rabani et al. 2011 (link); Siersbæk et al. 2014 (link)) with minor modifications. In short, mESCs were seeded in 2i medium in 150-mm dishes at 37°C and 5% CO2. Two days later, 500 μM 4-thiouridine (4sU, Sigma-Aldrich T4509) was added. Following 5-min incubation at 37°C, the medium was removed, and the labeling process was quenched by addition of TRIzol (Invitrogen 15596018) to mESCs. Spike-in RNAs (2.4 ng per million cells) were added, and samples were subjected to chloroform extraction and NaCl/isopropanol precipitation/ethanol washing before being resuspended in in DEPC-treated H2O. Two hundred fifty micrograms of extracted RNA was incubated with 500 μg EZ-Link HPDP-biotin (1 μg/μL in 100% dimethylformamide, Thermo Fisher Scientific 21341) to tag 4sU-labeled nascent RNA with biotin in biotinylating buffer (10 mM Tris at pH 7.5, 1 mM EDTA at pH 8.0) with rotation for 2 h at room temperature. RNA was cleaned up by chloroform:isoamyl alcohol extraction and precipitated with NaCl and isopropanol followed by a wash with ethanol and resuspension in DEPC-treated H2O. Next, to isolate 4sU-labled nascent RNA from total RNA, samples were incubated with 100 μL Dynabeads MyOne streptavidin T1 (Invitrogen 65601), washed twice with solution containing 100 mM NaOH and 50 mM NaCl and twice with 100 mM NaCl before use, and incubated for 15 min in streptavidin T1 in binding and washing buffer (5 mM Tris at pH 7.5, 0.5 mM EDTA at pH 8.0, 1 M NaCl, 0.1% Tween 20) while rotating at room temperature. After washing four times with binding and washing buffer, 4sU-labeled nascent RNA was released from biotin-streptavidin beads by elution in 100 mM DTT (Promega V3151) followed by purification with a NucleoSpin RNA kit (Macherey-Nagel 740955.250) according to the manufacturer's instructions.
Nascent RNA libraries were prepared using a NEBNext Ultra II directional RNA preparation kit (New England Biolabs E7760S) following the manufacturer's instructions. Briefly, nascent RNA was fragmentated into an average of 200 nt and reverse-transcribed into stranded DNA using random primers, which was followed by addition of sequencing bar-code during PCR amplification. Libraries were paired-end sequenced (40 cycles for each end) on a NextSeq 500 using an Illumina SE75Hi sequencing kit.
Pundhir S., Su J., Tapia M., Hansen A.M., Haile J.S., Hansen K, & Porse B.T. (2023). The impact of SWI/SNF and NuRD inactivation on gene expression is tightly coupled with levels of RNA polymerase II occupancy at promoters. Genome Research, 33(3), 332-345.
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3

Metabolic Labeling of Newly Synthesized RNA

Metabolic labelling of newly synthesized RNA was adapted from previously described protocols (29–31 (link)). In brief, cells were plated the day before to reach >90% of confluency before labelling. The nucleoside analogue 4-thiouridine (4sU) (Glentham Life Sciences, Cat# GN6085) was added to the cell culture medium at a final concentration of 500 μM for a 20-min pulse for mouse ES E14 and human U2OS cells. After the labelling period, the medium containing 4sU was removed, the cells were washed with ice cold 1× PBS and immediately lysed using TRI Reagent® (Molecular Research Center Inc., Cat# TR 188).
S. pombe cultures were grown to an OD600 of 0.8. Four-thiouracil (Sigma Aldrich, Cat# 440736) was freshly dissolved in DMSO and added to the cultures at a final concentration of 1 mM. Labelling was performed for 6 min. After this time period, yeast cells were pelleted, washed with ice-cold 1× PBS and aliquoted before being flash frozen in liquid nitrogen and stored at –80°C.
Fischer V., Hisler V., Scheer E., Lata E., Morlet B., Plassard D., Helmlinger D., Devys D., Tora L, & Vincent S.D. (2022). SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells. Nucleic Acids Research, 50(14), 7972-7990.
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4

Purification of Newly Synthesized RNA

The purification of newly synthesized RNA was based on previously described protocols starting from 4sU-labelled total RNA (29–31 (link)). As spike-in, 4sU-labelled S. pombe total RNA was added in a ratio 1:10 to labelled mESC total RNA preparations; or in a ratio of 1:25 to labelled U2OS total RNA preparations, to a final amount of 200 μg of total RNA prior to newly synthesized RNA purification. The RNA was precipitated and resuspended in 130 μl RNase-free water (Sigma Aldrich, Cat# 95284) and sonicated on a E220 Focused-ultrasonicator (Covaris) using the following settings: 1% duty factor, 100 W, 200 cycles per burst, 80 s to reach fragment size range from 10 kb to 200 bp. For purification, the fragmented total RNA was incubated for 10 min at 60°C and immediately chilled on ice for 2 min to open secondary RNA structures. The 4sU-labelled RNA was thiol-specific biotinylated by addition of 200 μg EZ-link HPDP-biotin (ThermoFisher Scientific, Cat# 21341), biotinylation buffer (10 mM HEPES–KOH pH 7.5 and 1 mM EDTA) and 20% DMSO (Sigma Aldrich, Cat# D8418). Biotinylation was carried out for 3 h at 24°C in the dark and with gentle agitation. After incubation, biotin excess was removed by adding an equal volume of chloroform and centrifugation at 16 000 g for 5 min at 4°C. RNA was precipitated from the aqueous phase by adding 0.1 volumes of 5 M NaCl and an equal volume of 100% isopropanol followed by centrifugation at 16 000 g for 30 min at 4°C. After washing with 75% ethanol, the RNA pellet was resuspended in 100 μl of RNase-free water and denatured for 10 min at 65°C followed by immediate chilling on ice for 5 min. The samples were incubated with 100 μl of streptavidin-coated μMACS magnetic beads (Miltenyi Biotec, Cat# 130-074-101) for 90 min at 24°C under gentle agitation. The μMACS columns (Miltenyi Biotec, Cat# 130-074-101) were placed on a MACS MultiStand (Miltenyi Biotec) and equilibrated with washing buffer (100 mM Tris–HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) before applying the samples twice on the columns. The columns were then washed one time with 600 μl, 700 μl, 800 μl, 900 μl and 1 ml washing buffer before elution of the newly synthesized RNA with two washes of 100 μl 0.1M DTT. The isolated newly synthesized RNA was recovered either using RNeasy MinElute Cleanup Kit (Qiagen, Cat# 74204) following manufacturer's instructions or by precipitation.
Fischer V., Hisler V., Scheer E., Lata E., Morlet B., Plassard D., Helmlinger D., Devys D., Tora L, & Vincent S.D. (2022). SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells. Nucleic Acids Research, 50(14), 7972-7990.
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5

4sU-labeled RNA measurement protocol

Total RNA was isolated using the miRNeasy kit (Qiagen) following the manufacturer's procedure. The protocol followed for the half-life measurement was performed as described previously (25 (link)) with few modifications. Briefly, 100 μg 4sU-labeled RNA were used for the biotinylation reaction. Biotinylation reactions were carried in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA pH 8.0) and 0.2 mg/ml EZ-Link Biotin-HPDP (Pierce) for 2 h at 25°C. Unbound Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1) extraction using MaXtract (high density) tubes (Qiagen), following the kit procedure. RNA was precipitated adding an equal volume of isopropanol and 1:10 volume of 5M NaCl; samples were then centrifuged 17 000 × g for 20 min. The pellet was washed with an equal volume of 75% ethanol and precipitated again at 17 000 × g for 10 min. The pellet was resuspended in 100 μl RNase-free water. Biotinylated RNA was captured using Dynabeads MyOne Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 100 μl Dynabeads with rotation for 15 min at 25°C. Beads were magnetically fixed and washed with 1 × Dynabeads washing buffer (5 mM Tris pH 7.5, 0.5 mM EDTA pH 8.0 and 1M NaCl). cDNA synthesis of the captured RNA-4sU was performed on beads using the SuperScript VILO cDNA synthesis kit (Life technologies), adding in the mastermix the RT probe for TaqMan® Assay for hsa-let-7b-5p (Life Technologies).
Lirussi L., Ayyildiz D., Liu Y., Montaldo N.P., Carracedo S., Aure M.R., Jobert L., Tekpli X., Touma J., Sauer T., Dalla E., Kristensen V.N., Geisler J., Piazza S., Tell G, & Nilsen H. (2022). A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours. Nucleic Acids Research, 50(18), 10449-10468.
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6

RNA Metabolic Labeling using 4sU

Metabolic labeling of newly synthesized RNA was adapted from previously described protocols (Rabani et al., 2011 (link); Rädle et al., 2013 ; Schwalb et al., 2016 (link)). In brief, the nucleoside analog 4-thiouridine (4sU) (Glentham Life Sciences, Cat#GN6085 or abcam, Cat#ab143718) was added to the cell culture medium at a final concentration of 500 μM for a 25 minutes pulse. After the labeling period, the medium containing 4sU was removed, the cells were washed with ice cold 1x PBS and immediately lysed using TRI® Reagent (Molecular Research Center Inc., Cat#TR 188). Total RNA was extracted following TRI® Reagent manufacturer’s instruction. To remove any potential genomic DNA contamination from the total RNA extracts, the TURBO DNA-free™ Kit (ThermoFisher Scientific, Cat#AM1907) was used following manufacturer’s instructions for rigorous DNase treatment.
To label Drosophila S2 cells, medium containing 4sU at a final concentration of 500 μM was added to the cells during 15 minutes under aluminum foil at room temperature. 4sU-containing medium was removed and 1xPBS was added to collect the cells using a cell scratcher. Cells were centrifuged, flash frozen in aliquots and stored at −80°C. For total RNA extraction, S2 cells were defrozen, lysed using TRI® Reagent (Molecular Research Center Inc., Cat#TR 188) and total RNA was isolated following manufacturer’s instruction.
Yeast cultures were grown to an OD600 of 0.8. 4-thiouracil (Sigma-Aldrich, Cat#440736) was freshly dissolved in DMSO and added to the cultures at a final concentration of 1 mM. Labeling was performed for 6 minutes. After this time period, yeast cells were pelleted, washed with ice-cold 1x PBS and aliquoted before being flash frozen and stored at −80°C. For total RNA extraction, the RiboPure™ RNA Purification Kit (ThermoFisher Scientific, Cat#AM1926) was used following manufacturer’s instruction.
Fischer V., Plassard D., Ye T., Reina-San-Martin B., Stierle M., Tora L, & Devys D. (2021). The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms. Cell reports, 36(8), 109598.
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7

RNA Metabolic Labeling using 4sU

Metabolic labeling of newly synthesized RNA was adapted from previously described protocols (Rabani et al., 2011 (link); Rädle et al., 2013 ; Schwalb et al., 2016 (link)). In brief, the nucleoside analog 4-thiouridine (4sU) (Glentham Life Sciences, Cat#GN6085 or abcam, Cat#ab143718) was added to the cell culture medium at a final concentration of 500 μM for a 25 minutes pulse. After the labeling period, the medium containing 4sU was removed, the cells were washed with ice cold 1x PBS and immediately lysed using TRI® Reagent (Molecular Research Center Inc., Cat#TR 188). Total RNA was extracted following TRI® Reagent manufacturer’s instruction. To remove any potential genomic DNA contamination from the total RNA extracts, the TURBO DNA-free™ Kit (ThermoFisher Scientific, Cat#AM1907) was used following manufacturer’s instructions for rigorous DNase treatment.
To label Drosophila S2 cells, medium containing 4sU at a final concentration of 500 μM was added to the cells during 15 minutes under aluminum foil at room temperature. 4sU-containing medium was removed and 1xPBS was added to collect the cells using a cell scratcher. Cells were centrifuged, flash frozen in aliquots and stored at −80°C. For total RNA extraction, S2 cells were defrozen, lysed using TRI® Reagent (Molecular Research Center Inc., Cat#TR 188) and total RNA was isolated following manufacturer’s instruction.
Yeast cultures were grown to an OD600 of 0.8. 4-thiouracil (Sigma-Aldrich, Cat#440736) was freshly dissolved in DMSO and added to the cultures at a final concentration of 1 mM. Labeling was performed for 6 minutes. After this time period, yeast cells were pelleted, washed with ice-cold 1x PBS and aliquoted before being flash frozen and stored at −80°C. For total RNA extraction, the RiboPure™ RNA Purification Kit (ThermoFisher Scientific, Cat#AM1926) was used following manufacturer’s instruction.
Fischer V., Plassard D., Ye T., Reina-San-Martin B., Stierle M., Tora L, & Devys D. (2021). The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms. Cell reports, 36(8), 109598.
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8

Purification of Newly Synthesized RNA

The purification of newly synthesized RNA was based on previously described protocols (Rabani et al., 2011 (link); Rädle et al., 2013 ; Schwalb et al., 2016 (link)). Labeled, total RNA of spike-in cells (D. melanogaster, S. cerevisiae or S. pombe) was added to labeled, total RNA from mouse ESCs in a ratio 1:5 to 1:10 prior to newly synthesized RNA purification to a final amount of 200–250 μg of total RNA. The RNA was precipitated and resuspended in 130 μL and sonicated using the following program on a Covaris E220 instrument: 1% duty factor, 100 W, 200 cycles per burst, 80 s. Fragment size ranged from 10 kb to 200 bp. For purification, the fragmented total RNA was incubated for 10 minutes at 60°C and immediately chilled on ice for 2 minutes to open secondary RNA structures. The 4sU-labeled RNA was thiol-specific biotinylated by addition of 200 μg EZ-link HPDP-biotin (ThermoFisher Scientific, Cat#21341), biotinylation buffer (10 mM HEPES-KOH pH 7.5 and 1 mM EDTA) and 20% DMSO (Sigma-Aldrich, Cat#D8418). Biotinylation was carried out for 3 hours at 24°C in the dark and with gentle agitations. After incubation, excess of biotin was removed by adding an equal volume of chloroform and centrifugation at 16,000 g for 5 minutes at 4°C. RNA was precipitated from the aqueous phase by adding 0.1 volumes of 5 M NaCl and an equal volume of 100% isopropanol followed by centrifugation at 16,000 g for 30 minutes at 4°C. After washing with 75% ethanol the RNA pellet was resuspended in 100 μL of RNase-free water and denatured for 10 minutes at 65°C followed by immediate chilling on ice for 5 minutes. The samples were incubated with 100 μL of streptavidin-coated μMACS magnetic beads (Miltenyi Biotec, Cat#130–074-101) for 90 minutes at 24°C under gentle agitations. The μMACS columns (Miltenyi Biotec, Cat#130–074-101) were placed on a MACS MultiStand (Miltenyi Biotec) and equilibrated with washing buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) before applying the samples twice to the columns. The columns were then washed one time with 600 μl, 700 μl, 800 μl, 900 μL and 1 mL washing buffer before eluting the newly synthesized RNA with two washes of 100 μL 0.1M DTT. The isolated newly synthesized RNA was recovered using RNeasy MinElute Cleanup Kit (QIAGEN, Cat#74204) following manufacturer’s instruction.
Fischer V., Plassard D., Ye T., Reina-San-Martin B., Stierle M., Tora L, & Devys D. (2021). The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms. Cell reports, 36(8), 109598.
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9

Purification of Newly Synthesized RNA

The purification of newly synthesized RNA was based on previously described protocols (Rabani et al., 2011 (link); Rädle et al., 2013 ; Schwalb et al., 2016 (link)). Labeled, total RNA of spike-in cells (D. melanogaster, S. cerevisiae or S. pombe) was added to labeled, total RNA from mouse ESCs in a ratio 1:5 to 1:10 prior to newly synthesized RNA purification to a final amount of 200–250 μg of total RNA. The RNA was precipitated and resuspended in 130 μL and sonicated using the following program on a Covaris E220 instrument: 1% duty factor, 100 W, 200 cycles per burst, 80 s. Fragment size ranged from 10 kb to 200 bp. For purification, the fragmented total RNA was incubated for 10 minutes at 60°C and immediately chilled on ice for 2 minutes to open secondary RNA structures. The 4sU-labeled RNA was thiol-specific biotinylated by addition of 200 μg EZ-link HPDP-biotin (ThermoFisher Scientific, Cat#21341), biotinylation buffer (10 mM HEPES-KOH pH 7.5 and 1 mM EDTA) and 20% DMSO (Sigma-Aldrich, Cat#D8418). Biotinylation was carried out for 3 hours at 24°C in the dark and with gentle agitations. After incubation, excess of biotin was removed by adding an equal volume of chloroform and centrifugation at 16,000 g for 5 minutes at 4°C. RNA was precipitated from the aqueous phase by adding 0.1 volumes of 5 M NaCl and an equal volume of 100% isopropanol followed by centrifugation at 16,000 g for 30 minutes at 4°C. After washing with 75% ethanol the RNA pellet was resuspended in 100 μL of RNase-free water and denatured for 10 minutes at 65°C followed by immediate chilling on ice for 5 minutes. The samples were incubated with 100 μL of streptavidin-coated μMACS magnetic beads (Miltenyi Biotec, Cat#130–074-101) for 90 minutes at 24°C under gentle agitations. The μMACS columns (Miltenyi Biotec, Cat#130–074-101) were placed on a MACS MultiStand (Miltenyi Biotec) and equilibrated with washing buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20) before applying the samples twice to the columns. The columns were then washed one time with 600 μl, 700 μl, 800 μl, 900 μL and 1 mL washing buffer before eluting the newly synthesized RNA with two washes of 100 μL 0.1M DTT. The isolated newly synthesized RNA was recovered using RNeasy MinElute Cleanup Kit (QIAGEN, Cat#74204) following manufacturer’s instruction.
Fischer V., Plassard D., Ye T., Reina-San-Martin B., Stierle M., Tora L, & Devys D. (2021). The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms. Cell reports, 36(8), 109598.
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10

4-sU RNA Isolation and Sequencing

4-sU (Sigma Aldrich, T4509)–labeled RNA isolation was performed as previously described (Rabani et al., 2011 (link)). Briefly, 300 µM 4-sU was added for 10 min to label confluent 36-h differentiated Scramble- and ShZc3h10-treated preadipocytes. Total RNA extraction was performed, as described above, and 1/50 of the total RNA was saved as input. The labeled RNA was isolated and processed as described (Austenaa et al., 2015 (link)). Briefly, the nascent 4-sU–labeled RNA was extracted from 50 µg of total TRIzol-isolated RNA, conjugated to N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (biotin-HPDP; Abcam), and precipitated with 50 µl streptavidin (MyOne Streptavidin T1, Invitrogen). 4-sU RNA samples were eluted in 20 µl of RNase-free water and analyzed by next-generation sequencing. Isolated nascent 4-sU–labeled RNA (20–30 ng) was used for cDNA library synthesis by using the QIAseq Stranded Total RNA Lib Kit (Qiagen, 180745) with no ribosomal depletion or polyA selection. Libraries were sequenced on an Illumina NextSeq 500.
Audano M., Pedretti S., Ligorio S., Gualdrini F., Polletti S., Russo M., Ghisletti S., Bean C., Crestani M., Caruso D., De Fabiani E, & Mitro N. (2021). Zc3h10 regulates adipogenesis by controlling translation and F-actin/mitochondria interaction. The Journal of Cell Biology, 220(3), e202003173.
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11

4SU-seq Protocol for Nascent RNA Capture

4SU-seq was performed essentially as described previously (Rabani et al. 2011 (link)). Briefly, 4-thiouridine (4SU; Sigma T4509) was added to ESCs in culture to a final concentration of 500 µM and incubated for 20 min at 37°C. Cells were harvested by trypsinization and washed twice with PBS at room temperature. Total RNA was isolated from 7 × 106 cells using Trizol according to the manufacturer's instructions (Invitrogen 15596026). Following precipitation, purified RNA was resuspended in 100 μL of RNase-free water and DNase-treated using the Turbo DNA-free kit according to the manufacturer's instructions (Invitrogen AM1907M). Residual inactivation beads were removed by spinning the RNA sample through a QIAshredder column at 1000g for 1 min (Qiagen). Two micrograms of total RNA input was retained for each sample and 30 μg was incubated for 1.5 h at room temperature with 60 μg of Biotin-HPDP (Pierce 21341; reconstituted in dimethylformamide at 1 mg/mL) in 1× biotinylation buffer (10 mM Tris at pH 7.4, 1 mM EDTA) to a total volume of 300 μL. Uncoupled biotin was removed through two consecutive rounds of 1:1 (v/v) chloroform extraction, followed by isopropanol/NaCl precipitation. RNA was resuspended in 100 μL of RNase-free water and mixed 1:1 (w/w) with µMacs Streptavidin beads (Miltenyi 130-074-101) and incubated for 15 min at room temperature with rotation. The RNA/bead mixture was applied to a µMacs column following pre-equilibration with wash buffer (100 mM Tris at pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20). The captured beads were then washed three times with 900 µL of 65°C wash buffer and three times with 900 µL of room temperature wash buffer. RNA was then eluted from the column by adding two consecutive rounds of 100 mM DTT. The eluate was added to 700 µL of buffer RLT (RNeasy MinElute cleanup kit; Qiagen 74204) and then purified according to the manufacturer's instructions. Prior to library preparation, ribosomal RNA was depleted from both the total and purified nascent RNA using the low-input RiboMinus eukaryote system v2 kit according to the manufacturer's instructions (Ambion A15027).
Libraries were constructed using the NEBNext Ultra II directional RNA library preparation kit for Illumina according to the protocol for ribosome-depleted RNA and with an 11-min RNA fragmentation step (NEB E7760). Library PCRs were supplemented with 2× SYBR dye (Sigma S9430) so that amplification could be monitored by quantitative PCR on a Roche LightCycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext multiplex oligos for Illumina, set 1, E7335) and amplified to linear phase. Size selection purifications following the ligation and amplification PCR steps were performed with 1× and 0.9× reaction volumes of Agencourt AMPure XP beads (Beckman Coulter A63880). Purified libraries were combined as an eight-sample equimolar pool containing the indexes 5–12 and sequenced on an Illumina NextSeq on a single high-output flow cell (paired-end 75-bp reads).
Boyle S., Flyamer I.M., Williamson I., Sengupta D., Bickmore W.A, & Illingworth R.S. (2020). A central role for canonical PRC1 in shaping the 3D nuclear landscape. Genes & Development, 34(13-14), 931-949.
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12

Classifying RNA Stability with Neural Network

Neural network was trained on the 5EU 24 h (Supplemental Tables S1, S2, samples 13–15) versus control (Supplemental Tables S1, S2, samples 10–12) sample parameters under the assumption that the 5EU 24 h sample solely contains labeled reads and the fact that the control sample solely contains unlabeled reads. Human RNA half-life distributions of previous studies (Rabani et al. 2011 (link); Schwalb et al. 2016 (link)) suggest that there is only a minor fraction of RNA species that live >24 h. Thus, the majority of RNA molecules in the 5EU 24 h samples (Supplemental Tables S1, S2, samples 13–15) are putatively 5EU-containing. Further, 24 h of labeling with 5EU has been shown to strongly label stable RNA species (Jao and Salic 2008 (link)). The trained neural network consists of eight dense layers with decreasing output shape (dense; units = 512, 265, 128, 64, 32, 16, 8, and 1; activation = “relu” and “sigmoid”; see below) with preceding batch normalization layers. Seven dropout layers (with 25% dropout) in between regularize the attempted classification. Training was conducted on 294,467 reads; validation was performed on 126,130 reads in 40 epochs with the R interface to Keras on a TensorFlow backend (https://github.com/rstudio/keras). For more detailed information on neural network design, please also see Supplemental Table S5 and the R code (see Software availability). The neural network was fivefold cross-validated with an accuracy of 0.87 and a FDR of 0.1 and was used to classify reads of the 5EU 60 min (Supplemental Tables S1, S2, samples 4–9) and 5EU 60 min heat shock (Supplemental Tables S1, S2, samples 16–20) samples into 5EU-labeled and unlabeled. A ROC analysis (1 – specificity vs. sensitivity) for all reads of the test set showed an AUC of 0.94. For reads with an alignment length >500 nt and >1000 nt, the AUC improved to 0.95 and 0.96. This suggests a small potential length bias. This is, however, uncritical to RNA stability assessment, especially on the RNA isoform level, as non-full-length reads are not considered. Note that different network architectures, such as additional dense layers, batch normalization, and dropout percentage, did not change the performance of the classification. The accuracy of the neural network is likely delimited by the lower accuracy of the direct RNA platform rather than the number of reads in the training set. This will very likely improve in the future owing to technical improvements driven by ONT. Note that the implemented multilayered data collection scheme (raw signal, base-calls, and alignment) might cause a high level of redundancy of the underlying data/information. Our neural network, however, is carefully designed, and we take a lot of computational measures to validate this (fivefold cross-validation) and thereby mitigate the risk of overtraining.
Maier K.C., Gressel S., Cramer P, & Schwalb B. (2020). Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms. Genome Research, 30(9), 1332-1344.
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13

4SU-seq for Transcriptome Dynamics

4SUseq was performed essentially as described previously (Rabani et al., 2011 (link)). Briefly, 4-thiouridine (4SU; Sigma, #T4509) was added to ESCs/NPCs in culture to a final concentration of 500 μM and incubated at 37°C for 20 min. Cells were harvested by trypsinisation and washed twice with PBS at r.t. Total RNA was isolated from 5x106 cells using Trizol according to the manufacturer’s instructions (Invitrogen, #15596026). Following precipitation, purified RNA was resuspended in 100 μL RNase-free water and DNase treated using the TURBO DNA-free kit according to the manufacturer’s instructions (Invitrogen, #AM1907M). Residual inactivation beads were removed by spinning the RNA sample through a QIAshredder column at 1000 g for 1 min (QIAGEN, #79654). Following quantification, 50 μg of RNA were incubated for 1.5 h at r.t. with 100 μg of Biotin-HPDP (Pierce, #21341; reconstituted in dimethylformamide at 1 mg/ml) in 1x Biotinylation Buffer (10 mM Tris pH 7.4 and 1 mM EDTA) to a total volume of 500 μl. Uncoupled biotin was removed through two consecutive rounds of 1:1 v/v chloroform extraction followed by isopropanol/NaCl precipitation. RNA was resuspended in 100 μL of RNase free water and mixed 1:1 w/w with μMacs Streptavidin beads (Miltenyi, #130-074-101) and incubated for 15 min at RT with rotation. The RNA / bead mixture was applied to a μMacs column following pre-equilibration with wash buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). The captured beads were then washed with 3 × 900 μl of 65°C wash buffer and 3 × 900 μl RT wash buffer. RNA was then eluted from the column by adding two consecutive rounds of 100 mM DTT. The eluate was added to 700 μl Buffer RLT (RNeasy MinElute Cleanup Kit; QIAGEN, #74204) and then purified according to the manufacturer’s instructions. Prior to library preparation, ribosomal RNA was depleted from the purified 4SU incorporate RNA using the low Input RiboMinus Eukaryote System v2 kit according to the manufacturer’s instructions (Ambion, # A15027).
Sequencing libraries were constructed using the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina according to the protocol for ribosome depleted RNA and with a 10 min RNA fragmentation step (NEB, #E7760). Library PCRs were supplemented with 2x SYBR dye (Sigma, #S9430) so that amplification could be monitored by quantitative PCR on a Roche lightcycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext Multiplex Oligos for Illumina - Set 1. #E7335) and amplified to linear phase. Libraries were purified and then combined into 4 sample equimolar pools containing the indexes 1, 3, 6 and 8.
Benabdallah N.S., Williamson I., Illingworth R.S., Kane L., Boyle S., Sengupta D., Grimes G.R., Therizols P, & Bickmore W.A. (2019). Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation. Molecular Cell, 76(3), 473-484.e7.
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14

4sU-RNA-seq Protocol for Transcriptome Analysis

4sU-RNA-seq was performed as described in Rabani et al.60 (link). In brief, cells were incubated with the medium supplemented with 500 μM 4sU for 15 min, after which medium was rapidly removed and cells were lysed with 5 mL of Trizol reagent (Life Technologies). Total RNA was treated with Ambion DNA-free DNase Treatment kit (Life Technologies) and resuspended in water. For each μg of total RNA, 2 μL of Biotin-HPDP (Pierce, 50 mg EZ-Link Biotin-HPDP) dissolved in DMF at a concentration of 1 mg/mL, and 1 μL of 10X Biotinylation buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA), was added. After incubation for 15 min at 25 °C, the RNA was purified using two rounds of chloroform purification in Phase Lock Gel Heavy Tubes (Eppendorf), precipitation with an equal volume of isopropanol and 1/10 volume of 5 M NaCl, and finally resuspended in water.
Biotinylated 4sU-RNA was recovered using the μMacs Streptavidin Kit (Miltenyi). Per microgram of recovered biotinylated 4sU-RNA, 0.5 μL of streptavidin beads were added, in a total volume of 200 μL. Samples were washed six times, eluted in fresh 100 mM DTT, and further purified using the RNA Clean & Concentrator-5 kit (Zymoresearch). Libraries for RNA-seq were constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®.
Zhang T., Wei G., Millard C.J., Fischer R., Konietzny R., Kessler B.M., Schwabe J.W, & Brockdorff N. (2018). A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes. Nature Communications, 9, 3798.
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15

Metabolic Labeling and Isolation of Newly Synthesized RNAs

MCF7 and T47D cells (106) were labeling with 4-thiouridine (4sU) at 250 μM for 45 minutes at the treatment conditions defined in Figure 8A. The dose and duration used was based on publications reporting that this condition produces a sufficient level of labeling and without noticeable interference with RNA synthesis in mammalian cells [66 (link), 68 (link), 69 (link)]. RNA was isolated using miRNeasy MinElute; 4sU-labeled (newly synthesized) RNA in a total of 20 μg RNA was biotinylated using the EZ-Link Biotin-HPDP kit (Thermo Fisher Scientific) according the manufacturer’s protocol. Briefly, RNA was heated at 65°C and cooled on ice, followed by incubation with EZ-link Biotin HPDP (2 mg/ml dissolved in dimethylformamide/DMF) at room temperature for 2 hours in a labeling buffer (10mM Tris pH 7.4, 1 mM EDTA). Unbound Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1) extraction. RNA was precipitated using isopropanol and washed with 75% ethanol. GlycoBlue Coprecipitant (Life Tech, AM9516), 15 μg was used as an RNA indicator. RNA was heated at 65°C. Biotinylated RNA was captured using conjugated MyOne Streptavidin C1 Beads (20 μl) (Invitrogen), which was prepared according to the manufacturer’s instruction, at room temperature for 90 minutes. The streptavidin C1 beads were then washed using a buffer (10 mM TrisCl, pH7.5; 1 mM EDTA; 1M NaCl) at 65°C for 1–2 minutes three times. The unlabeled RNA was collected by pooling supernatants and three washes. The beads were further washed at room temperature twice. The biotin-labelled RNAs were eluded using 100 mM dithiothreitol (DTT) at room temperature twice. The unlabeled RNAs and biotin-labelled RNAs were purified using RNeasy MiniElute Kits (Qiagen). RNAs were then used for RT and qPCR experiments.
Wei F., Hao P., Zhang X., Hu H., Jiang D., Yin A., Wen L., Zheng L., He J.Z., Mei W., Zeng H, & Tang D. (2018). Etoposide-induced DNA damage affects multiple cellular pathways in addition to DNA damage response. Oncotarget, 9(35), 24122-24139.
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16

Isolation and Purification of 4sU-Labeled RNA

4sU-RNA was generated and isolated essentially as described in (Rabani et al., 2011 (link)). In detail, 4-thiouridine (4sU, Sigma, T4509) was dissolved in sterile PBS and stored at −20°C. 4sU was thawed just before use and added to the cells in the growing media at a concentration of 500 μM. Cells were incubated with the 4sU-supplemented medium for 12 min. Cell culture medium was rapidly removed from cells and 5 mL of Trizol reagent (Life Technologies) was added. Total RNA was extracted as described above, treated with DNase using the Ambion DNA-free DNase Treatment kit (Life Technologies) according to the manufacturer’s instructions, and resuspended in water. For each μg of total RNA, 2 μL of Biotin-HPDP (Pierce, 50mg EZ-Link Biotin-HPDP), previously dissolved in DMF at a concentration of 1 mg/mL, and 1 μL of 10xBiotinylation buffer (100 mM Tris HCl pH 7.4, 10 mM EDTA), was added. The reaction was incubated with rotation for 15 min at 25°C. RNA was transferred to Phase Lock Gel Heavy Tubes (Eppendorf), and an equal volume of chloroform was added. After vigorously mixing, tubes were left incubating for 3 min at 25°C and then centrifuged at 13,000rpm for 5 min at 4°C. The upper phase was transferred to new Phase Lock Gel Heavy Tubes, and chloroform added again. After further centrifugation, the upper phase was transferred to a tube containing an equal volume of isopropanol and 1/10 volume of 5 M NaCl. After inversion, the tubes were centrifuged at 13,000 rpm for 20 min at 4°C. Pellet was washed in 75% ethanol and resuspended in water.
Biotinylated 4sU-RNA was recovered using the μMacs Streptavidin Kit (Miltenyi), with a modified protocol. Per μg of recovered biotynilated 4sU-RNA, 0.5 μL of streptavidin beads were added, in a total volume of 200 μL. Samples were incubated with rotation for 15 min at 25°C. μMacs columns supplied with the μMacs Streptavidin Kit were equilibrated in 1 mL of washing buffer (100 mM Tris HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20) at 65°C. Samples were added to the columns that were then washed 6 times with washing buffer, 3 times at 65°C and three times at 25°C. RNA was eluted in freshly-prepared 100 mM DTT. RNA was further purified using the RNA Mini Clean & Concentrator kit (Zymoresearch) according to the manufacturer’s guidelines. 1 μL of 4sU-labeled RNA was quality-checked using the Agilent RNA 6000 Pico kit (Agilent technologies) according to the manufacturer’s instructions, and run on a 2100 Bioanalyzer Instrument (Agilent).
Pintacuda G., Wei G., Roustan C., Kirmizitas B.A., Solcan N., Cerase A., Castello A., Mohammed S., Moindrot B., Nesterova T.B, & Brockdorff N. (2017). hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing. Molecular Cell, 68(5), 955-969.e10.
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17

Measurement of mRNA Stability and Synthesis

Beas2B.tTA cells were plated in 15 cm dishes with 30 ml medium 1 day before labeling and were ∼80% confluent when 4sU was added. To prepare a 40 mM 4sU stock solution, 100 mg of 4sU (Sigma, T4509) was dissolved in 9.6 ml phosphate buffered saline (PBS) and used immediately. To monitor mRNA decay (19 (link)), 300 μl of 4sU stock solution was added to each plate and mixed into the medium by gentle shaking followed by a 4 h incubation. After washing cells twice with 25 ml PBS (pre-warmed to 37°C), cells were harvested immediately (t0) or maintained in an incubator for 4 h (t4). To measure newly synthesized transcripts, 375 μl of 4sU stock solution was added to each plate; after a 20 min incubation cells were washed with 20 ml ice-cold PBS and then harvested in Buffer RLT for total RNA extraction with the RNeasy mini kit.
The 4sU labeled RNAs in the total RNA isolates were biotinylated with the following procedure. A 50 mg EZ-Link Biotin-HPDP (Pierce, 21341) was dissolved in 50 ml of dimethylformamide (DMF) to produce 1 mg/ml biotin stock solution. The labeling reaction was carried out in siliconized 15 ml tubes containing 300 μl 10× TE buffer (100 mM Tris pH7.4 + 10 mM ethylenediaminetetraacetic acid (EDTA)), 600 μl biotin stock solution, 300–400 μg total RNA and water to 3 ml. The tubes were kept in the dark and rotated for 2 h. To remove free chemicals, an equal volume (3 ml) of chloroform/isoamylalcohol (24:1) was added to the reaction and hand shaken vigorously to homogenize the mixture. After transfer to MaXtract high density tubes (Qiagen), hand shaking to produce a homogenous suspension, waiting for 2–3 min and then spinning at 1500 g for 6 min at 4°C, the upper aqueous phase was transferred to multiple 1.7 ml tubes. After addition of 1/10 volume of 5 M NaCl and one volume of isopropanol, tubes were inverted to mix thoroughly and incubated at room temperature for 10 min and at −20°C for 20 min. After spinning at 20 000 × g for 30 min at 4°C, removing the supernatant, adding one volume of 75% ethanol and spinning at 20 000 × g for 15 min at 4°C, the supernatant was removed and briefly air-dried. Pellets were re-dissolved in 50 μl water, combined and stored at −80°C.
Biotin labeled 4sU RNA was isolated with the following procedure. A total of 300 μg total RNA containing biotinlylated RNAs was mixed with 10× TE buffer and water to produce a final RNA concentration of ∼100 μg/μl, followed by heating at 65°C for 10 min and immediately placing tubes on ice for 5 min. Next, we used μMACS streptavidin beads and columns (Miltenyi, 130–074-101) to isolate 4sU RNAs according to the manufacturer’s instructions. Final 4sU RNA elutes were purified with RNeasy MinElute Spin columns (Qiagen, 74204) in RNase free water. The resulting RNAs were reverse transcribed into cDNA with oligo-dT primer and used for qRT-PCR. GAPDH mRNA was used for normalization. We quantified chemokine mRNA stability by determining the relative fraction of RNA remaining after 4 h: ([chemokine RNA at 4 h]/[GAPDH at 4 h])/([chemokine RNA at 0 h]/[GAPDH at 0 h]). We quantified newly synthesized chemokine mRNA as the ratio of ([chemokine RNA after 20 min pulse]/[GAPDH RNA after 20 min pulse])/([mean wild-type chemokine RNA after 20 min pulse]/[mean wild-type GAPDH RNA after 20 min pulse]).
Zhao W., Siegel D., Biton A., Tonqueze O.L., Zaitlen N., Ahituv N, & Erle D.J. (2017). CRISPR–Cas9-mediated functional dissection of 3′-UTRs. Nucleic Acids Research, 45(18), 10800-10810.
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18

Quantifying Newly Synthesized RNA

4sU (Sigma catalog no. T4509) was added to culture medium at a final concentration of 300 μM. Cells were pulsed for 3 h and subsequently chased with 1 mM uridine (Sigma catalog no U3003). For continuous labeling experiments, cells were treated with 4sU (300 μM) for increasing lengths of time (15 min, 30 min, 45 min, 60 min, 120 min, 180 min). Total RNA was extracted at each time point by the miRNeasy mini kit (Qiagen catalog no. 217004), according to the manufacturer's instructions, including the recommended DNase I digestion. Separation of 4sU-labeled newly synthesized RNA from nonlabeled pre-existing RNA was performed as previously described (Rabani et al. 2011 (link)) with minor modifications (see Supplemental Material).
Marzi M.J., Ghini F., Cerruti B., de Pretis S., Bonetti P., Giacomelli C., Gorski M.M., Kress T., Pelizzola M., Muller H., Amati B, & Nicassio F. (2016). Degradation dynamics of microRNAs revealed by a novel pulse-chase approach. Genome Research, 26(4), 554-565.
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19

Transcriptional Dynamics in Breast Cells

We isolated total RNA, newly transcribed RNA and preexisting RNA from MCF10A cells that were metabolically pre-labeled using 4sU. The isolated RNA was quantified using Fluidigm and qPCR. For labeling, cells were treated for 20, 40, 60, 120 and 240 min with a combination of 4sU and EGF, or 4sU only. Because a short labeling of 10 min is sufficient for 4sU uptake (35 (link),38 (link)), the fraction of unlabeled EGF-responsive genes was negligible in our studies. Differences in the amount of total RNA were corrected by normalizing to the levels of two housekeeping genes (G6PD and PGK1).
Enuka Y., Lauriola M., Feldman M.E., Sas-Chen A., Ulitsky I, & Yarden Y. (2015). Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Research, 44(3), 1370-1383.
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20

Biotinylation and Purification of 4sU RNA

Biotinylation and purification of 4sU labeled RNA was done as described in [42 (link)], with minor changes. A total of 100 to 180 μg RNA was used for the biotinylation reaction. 4sU-labeled RNA was biotinylated using EZ-Link Biotin-HPDP (Pierce), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/mL and stored at -80°C. Biotinylation was done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP for 2 h with rotation at 25°C. Unbound Biotin-HPDP was removed by chloroform/isoamylalcohol (24:1) extraction using MaXtract (high density) tubes (Qiagen). RNA was precipitated at 20,000 g for 20 min at 4°C with a 1:10 volume of 5M NaCl and an equal volume of isopropanol. The pellet was washed with an equal volume of 75% ethanol and precipitated again at 20,000 g for 10 min at 4°C. The pellet was left to dry followed by resuspension in 100 μL RNase-free water. Biotinylated RNA was captured using Dynabeads MyOne Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 100 μL Dynabeads with rotation for 15 min at 25°C. Beads were magnetically fixed and washed with 1× Dynabeads washing buffer. RNA-4sU was eluted with 100 μL of freshly prepared 100 mM dithiothreitol (DTT), and cleaned on RNeasy MinElute Spin columns (Qiagen).
Fuchs G., Voichek Y., Benjamin S., Gilad S., Amit I, & Oren M. (2014). 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biology, 15(5), R69.
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21

Labeling and Purifying 4sU-Labeled RNA

Cells were seeded in 15-cm plates for 24 h. 4-thiouridine (4sU, Sigma) was added to the cell-culture medium to a 500-μM final concentration 15 min before RNA extraction. We used this time point, assuming that there was negligible degradation of RNA. Biotinylation and purification of 4sU-labeled RNA was based on the protocol used by Rabani et al.17 (link). A certain amount (usually 10%) of RNA was set aside as total RNAs before biotinylated RNA was captured with Dynabeads.
Zheng G.X., Do B.T., Webster D.E., Khavari P.A, & Chang H.Y. (2014). Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs. Nature structural & molecular biology, 21(7), 585-590.
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22

Optimized PAR-CLIP for Studying Non-Canonical RNA-Binding Proteins

For CLIP experiments, ESCs were crosslinked with 400 mJ/cm2 UVC (254 nm) and then processed as described below.
For PAR-CLIP, ESCs were grown in standard conditions and pulsed with 500 μM 4-SU (Sigma) for 2 h. In some experiments the 4-SU was chased with 500 μM uridine. After washing the plates with PBS, RNA–protein crosslinks were generated by irradiating 4-SU-treated cells with 400 mJ/cm2 UVB (312 nm) using a Stratalinker UV crosslinker (Stratagene, CA). These conditions are different than those previously reported46 and were optimized to maximize crosslinking of RNA to EZH2 (Fig. 1b), a non-canonical RNA-binding protein. Although Rabani et al. observed 5′-biased 4-SU incorporation at the Ifih1 gene after a 45 minute pulse47 (link), it was only at the first time point after gene induction, suggesting that the 5′ bias reflected the increased rate of initiation events rather than a predisposition of RNAPII to incorporate 4-SU at the beginning of transcriptional units. Indeed, in the same experiment, later time points showed homogeneous 4-SU incorporation across the body of the same transcript47 (link). Incubation with 4-SU for 2 h was previously shown to label RNA efficiently in a broad range of cells48 (link) and is considerably longer than the half-life of a majority of mature mRNAs and virtually all pre-mRNAs47 (link). These optimized PAR-CLIP conditions may be useful to determine contacts with noncoding RNAs for other non-canonical RNA-binding proteins found in chromatin.
Whole cell extracts were obtained by incubating the cells for 10 min at 37°C in an appropriate volume of CLIP buffer (20 mM HEPES pH 7.4, 5 mM EDTA, 150 mM NaCl, 2% lauryldimethylbetaine) supplemented with protease inhibitors, 20 U/ml Turbo DNase (Life technologies), and 200 U/ml murine RNase inhibitor (New England Biolabs) and lysates were cleared by centrifugation. Prior to immunoprecipitations, epitope-tagged EZH2 was affinity-purified by using Strep-Tactin beads (IBA) and eluted with CLIP buffer containing 2 mM biotin (Sigma). Immunoprecipitations were carried out with ChIP-grade anti-HA antibody (abcam) in the same CLIP buffer for 16 hours at 4°C, after which, when required, the extracts were treated with various concentration of RNAse A + T1 cocktail (Ambion) for 5′ at 37°C. Immunocomplexes were recovered by adding protein G-coupled dynabeads (Life technologies) for 45 min at 4°C. Contaminating DNA was removed by treating the beads with Turbo DNase (2U in 20 μl). Crosslinked RNA was labeled by successive incubation with 5U Antarctic phosphatase (New England Biolabs) and 5U T4 PNK (New England Biolabs) in presence of 10 μCi [γ-32P] ATP (PerkinElmer, MA). Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for 1–24 hours.
For PAR-CLIP-seq experiments, 3′-blocked DNA adapter (100 pmol/μl) was ligated to the RNA after dephosphorylation and before 5′ 32P end-labeling by incubating the beads with T4 RNA ligase 1 (New England Biolabs) for 1 hour at 25°C. Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for ∼4 hours. Bands of interest were excised and the RNA eluted from the membrane by treating with proteinase K (Roche, 4 mg/ml) for 30′ at 37°C and then proteinase K in presence of 3.5M urea for 30′ at 55°C. After phenol/chloroform extraction, custom designed 5′ RNA adapters were ligated, the products size-selected on polyacrylamide gels, and libraries amplified and sequenced on an Illumina HiSeq 2000 sequencing system.
Kaneko S., Son J., Shen S.S., Reinberg D, & Bonasio R. (2013). PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells. Nature structural & molecular biology, 20(11), 1258-1264.
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23

Optimized PAR-CLIP for Studying Non-Canonical RNA-Binding Proteins

For CLIP experiments, ESCs were crosslinked with 400 mJ/cm2 UVC (254 nm) and then processed as described below.
For PAR-CLIP, ESCs were grown in standard conditions and pulsed with 500 μM 4-SU (Sigma) for 2 h. In some experiments the 4-SU was chased with 500 μM uridine. After washing the plates with PBS, RNA–protein crosslinks were generated by irradiating 4-SU-treated cells with 400 mJ/cm2 UVB (312 nm) using a Stratalinker UV crosslinker (Stratagene, CA). These conditions are different than those previously reported46 and were optimized to maximize crosslinking of RNA to EZH2 (Fig. 1b), a non-canonical RNA-binding protein. Although Rabani et al. observed 5′-biased 4-SU incorporation at the Ifih1 gene after a 45 minute pulse47 (link), it was only at the first time point after gene induction, suggesting that the 5′ bias reflected the increased rate of initiation events rather than a predisposition of RNAPII to incorporate 4-SU at the beginning of transcriptional units. Indeed, in the same experiment, later time points showed homogeneous 4-SU incorporation across the body of the same transcript47 (link). Incubation with 4-SU for 2 h was previously shown to label RNA efficiently in a broad range of cells48 (link) and is considerably longer than the half-life of a majority of mature mRNAs and virtually all pre-mRNAs47 (link). These optimized PAR-CLIP conditions may be useful to determine contacts with noncoding RNAs for other non-canonical RNA-binding proteins found in chromatin.
Whole cell extracts were obtained by incubating the cells for 10 min at 37°C in an appropriate volume of CLIP buffer (20 mM HEPES pH 7.4, 5 mM EDTA, 150 mM NaCl, 2% lauryldimethylbetaine) supplemented with protease inhibitors, 20 U/ml Turbo DNase (Life technologies), and 200 U/ml murine RNase inhibitor (New England Biolabs) and lysates were cleared by centrifugation. Prior to immunoprecipitations, epitope-tagged EZH2 was affinity-purified by using Strep-Tactin beads (IBA) and eluted with CLIP buffer containing 2 mM biotin (Sigma). Immunoprecipitations were carried out with ChIP-grade anti-HA antibody (abcam) in the same CLIP buffer for 16 hours at 4°C, after which, when required, the extracts were treated with various concentration of RNAse A + T1 cocktail (Ambion) for 5′ at 37°C. Immunocomplexes were recovered by adding protein G-coupled dynabeads (Life technologies) for 45 min at 4°C. Contaminating DNA was removed by treating the beads with Turbo DNase (2U in 20 μl). Crosslinked RNA was labeled by successive incubation with 5U Antarctic phosphatase (New England Biolabs) and 5U T4 PNK (New England Biolabs) in presence of 10 μCi [γ-32P] ATP (PerkinElmer, MA). Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for 1–24 hours.
For PAR-CLIP-seq experiments, 3′-blocked DNA adapter (100 pmol/μl) was ligated to the RNA after dephosphorylation and before 5′ 32P end-labeling by incubating the beads with T4 RNA ligase 1 (New England Biolabs) for 1 hour at 25°C. Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for ∼4 hours. Bands of interest were excised and the RNA eluted from the membrane by treating with proteinase K (Roche, 4 mg/ml) for 30′ at 37°C and then proteinase K in presence of 3.5M urea for 30′ at 55°C. After phenol/chloroform extraction, custom designed 5′ RNA adapters were ligated, the products size-selected on polyacrylamide gels, and libraries amplified and sequenced on an Illumina HiSeq 2000 sequencing system.
Kaneko S., Son J., Shen S.S., Reinberg D, & Bonasio R. (2013). PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells. Nature structural & molecular biology, 20(11), 1258-1264.
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Variable analysis

independent variables
  • Time after LPS stimulation
dependent variables
  • Total RNA quantity
  • RNA quality (tested on Bioanalyzer)
  • 4sU-labeled RNA quantity
control variables
  • Total RNA input (20 μg) for biotinylation reaction
  • Biotinylation reaction conditions (labeling buffer, Biotin-HPDP concentration, duration, temperature)
  • RNA precipitation and washing conditions
  • Biotinylated RNA capture and washing conditions
positive controls
  • None specified
negative controls
  • None specified

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