GSM6738180: HEK293CDK7as_TTseq_TotalRNA_1-NM-PP1_15min_Rep2; Homo sapiens; RNA-Seq
TT-seq: Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026), 4sU-labeled RNA was extracted according to Gressel at al., protocols.io, 2019. mNET-seq: mNET-seq was performed as previously described (Caizzi et al. 2021; Gressel et al. 2017; Nojima et al. 2016; Schlackow et al. 2017) with minor changes. 50 mio CDK7as cells were treated for 15 or 30 min with 7.5 μM of 1-NM-PP1 or DMSO (solvent control). After the treatment, chromatin isolation was performed as described in the protocol for cellular fractionation (Conrad et al. 2017) using 25 mio cells per reaction. All buffers were supplemented with 7.5 μM 1-NM-PP1 or respective amounts of DMSO, protease (Sigma-Aldrich, P8340), and phosphatase (Roche, 4906837001) inhibitors. After the fractionation, the chromatin pellet was digested in 100 μl MNase digest buffer (1x MNase buffer, 1x BSA, 100 mM NaCl, 50 units/μl MNase (NEB, M0247S)) at 37°C and 1,400 rpm for 2 min. The digestion was stopped by adding 11 μl of 250 mM EGTA, and samples were centrifuged at 13,000 g for 5 min at 4°C. Supernatants were pooled together for each sample and 8-fold diluted with IP buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, 0.3% empigen (Sigma-Aldrich, 30326)). 15 μg of anti-Pol II CTD antibody (Diagenode, C15200004) conjugated with 125 μl of Dynabeads M-280 Sheep Anti-Mouse IgG (Invitrogen, 11201D) were added to each sample and incubated on a rotating wheel at 4°C and 12 rpm for 1 h. After IP, beads were washed seven times with 1 ml of IP buffer and once with 300 μl of PNKT buffer (1x T4 PNK buffer (NEB, M0236L), 0.1% Tween-20). After washes, 5' RNA ends were phosphorylated with T4 Polynucleotide Kinase (PNK) (3' phosphatase minus; NEB, M0236L) at 800 rpm for 10 min at 37°C. Next, beads were washed once with IP buffer, and 0.5 ml of TRIzol reagent (Invitrogen, 15596026) was added to the beads. 2.5 ng of S. cerevisiae spike-ins were added to each sample in TRIzol. RNA spike-ins for mNET-seq were obtained from S. cerevisiae cells as previously described (Caizzi et al. 2021). RNA was extracted from beads, followed by RNA precipitation with 100% ethanol and GlycoBlue coprecipitant (Invitrogen, AM9515) at -80°C overnight. RNA pellets were washed twice with 70% ethanol, air-dried, and dissolved in 7 M urea. In order to size select 25-110 nt RNA, samples were resolved on 6% polyacrylamide gel containing 7 M urea. RNA of corresponding sizes was extracted from the gel and precipitated with 100% ethanol and GlycoBlue coprecipitant. MNase-seq: 5 mio CDK7as cells were treated for 15 min with 7.5 μM of 1-NM-PP1 or DMSO (solvent control) and centrifuged at 500 g for 3 min at 4°C. Cell pellets were resuspended in 50 μl of Buffer I (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 10 % sucrose, 0.5 mM CaCl2) and incubated at 37°C for 2 min. Next, we added 500 μl of Buffer II (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 10 % sucrose, 2 mM CaCl2) supplemented with 3,000 U of MNase (NEB, M0247S) and incubated at 37°C for 9 min. MNase digestion was quenched with 11 μl of EGTA (500 mM). 550 μl of SDS Lysis Buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) was added, and samples were incubated on ice for 10 min. Samples were then treated with 0.1 μg/μl RNAse A (Thermo Scientific, EN0531) for 1.5 h at 37°C and 0.05 μg/μl Proteinase K (Life Technologies, AM2546) for 4 h at 65°C. DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, P2069), followed by DNA precipitation with an equal volume of 100% ethanol and 200 mM NaCl for 2 h at -20°C. DNA pellets were washed twice with 1 ml of 70% ethanol, air-dried, and dissolved in water. MNase-ChIP-seq: MNase-ChIP-seq was performed as previously described (Skene et al. 2015) with the following changes. 40 mio CDK7as cells were treated for 30 min with 7.5 μM of 1-NM-PP1 or DMSO (solvent control), following 8 min crosslinking with 1% formaldehyde (16% formaldehyde (w/v), methanol-free, Thermo Fisher Scientific, 28908). The crosslinking was quenched for 5 min with 125 mM glycine, and cells were washed with 10 ml PBS at room temperature. 2 ml of ice-cold PBS was added to plates for cell scraping. Scraped cells were transferred to falcons, washed twice with 5 ml ice-cold PBS, and centrifuged at 200 g for 5 min at 4°C. All buffers were supplemented with protease (Sigma-Aldrich, P8340) and phosphatase (Roche, 4906837001) inhibitors. Cell pellets were lysed with 5 ml of Farnham Lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% NP-40) for 10 min on ice. Nuclei were pelleted by centrifugation at 1,700 g for 5 min at 4°C and washed with ice-cold PBS. Nuclei pellets were resuspended in 200 μl of 1% SDS Lysis buffer (50 mM Tris HCl pH 8.0, 10 mM EDTA, 1% SDS) and incubated for 10 min at room temperature to permeabilize nuclei. The reaction was quenched with 1.8 ml of IP buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with 5 mM CaCl2 and incubated for 2 min at 37°C before the addition of 16,000U of MNase (NEB, M0247S) for 20 min at 37°C. MNase digestion was quenched with 40 μl of EDTA (500 mM) and 20 μl EGTA (500 mM). 1 ml of sample was transferred to AFA milliTubes (Covaris, 520135) and sonicated with a Covaris S220 Focused-ultrasonicator with the following parameters: 5% duty cycle, 140 W peak incident power, 200 cycles per burst, 600 sec processing time, 6°C bath temperature, continuous degassing mode, water level 8. Sonicated samples were centrifuged at 10,000 g for 15 min at 4°C, and supernatants were snap-frozen in liquid nitrogen and stored at −80°C before use. For each IP, 30 μg of sample chromatin and 200 ng of Drosophila S2 MNase-digested crosslinked chromatin were diluted in IP buffer to obtain a final concentration of 0.05% SDS and a final volume of 1.6 ml. Pre-clearing was carried out by adding 20 μl of Dynabeads Protein G (Thermo Fisher Scientific, 10009D) diluted in IP buffer, and incubating for 30 min at 4°C on the rotation wheel. Pre-cleared samples were incubated with 10 μg of Pol II (RPB1, Diagenode, C15200004), 10 μg of Ser5P (phospho-CTD Ser-5 of RPB1, Merck Millipore, 04-1572), 8 μl of Mediator (MED26, Cell Signaling, 14950), 6 μg of TFIIB (Santa Cruz, sc-271736), 5 μg of TFIIE (TFIIE-α, abcam, ab28177), 6 μg of NELF (NELF-E, Bethyl Laboratories, A301-913A) or 6 μg of SPT5 (Proteintech, 16511-1-AP) together with 1 μg of Spike-in (Active Motif, 61686) antibodies at 4°C overnight. The next day samples were centrifuged at 16,000g for 2 min at 4°C and inputs were taken for each sample. Samples were incubated for 2 h at 4°C with Dynabeads Protein G. Beads were washed once with Buffer 1 (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), four times with Buffer 2 (20 mM Tris HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Buffer 3 (10 mM Tris HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium-deoxycholate), and three times with TE buffer (10 mM Tris HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). After the last wash, 200 μl of elution buffer (0.1 M NaHCO3, 160 mM NaCl, 1% SDS) was added to the beads, and samples were de-crosslinked overnight at 65°C. Next, samples were diluted with 200 μl of PBS and treated with 0.02 μg/μl RNAse A (Thermo Scientific, EN0531) for 1.5 h at 37°C and 0.2 μg/μl Proteinase K (Life Technologies, AM2546) for 2 h at 45°C. DNA was extracted with an equal volume (400 μl) of phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, P2069), followed by DNA precipitation with 100% ethanol and 200 mM NaCl for 1 h at -80°C. DNA pellets were washed twice with 70% ethanol, air-dried, and dissolved in water. TT-seq: Libraries were prepared from 100 ng of RNA with the strand-specific Ovation Universal RNA-Seq library kit (NuGEN, 0343-32). mNET-seq: 8.4 ng of RNA for replicate 1 and 12.3 ng of RNA for replicate 2 were used for library preparation with TruSeq Small RNA Library Prep Kit (RS-200-0048) according to the manufacturer's instructions. MNase-seq: Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) according to the manufacturer's instructions. MNase-ChiP-seq: Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) according to the manufacturer's instructions with adjusted for short fragments end repair, 5' phosphorylation, and dA-tailing thermocycler program (30 min at 20°C, 1 h at 50°C; the heated lit set to 60°C). After amplification, libraries were double-sided (1.0 – 1.6x) size-selected with Agencourt AMPure XP beads (Beckman Coulter, A63881).