Cell cultureHCT116 KAP1dTAG and HCT116 parental cells (to build the cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog 11965118) supplemented with 7% Fetal Bovine Serum (FBS) (MilliporeSigma, catalog F4135) and 1% Penicillin/Streptomycin (P/S) (Gibco, catalog 15140163) at 37 °C with 5% CO2. The original HCT116 parental cell line was purchased from ATCC (catalog CCL-247). Mycoplasma tests (SouthernBiotech, catalog 13100-01) were conducted every ~3–6 months.dTAG and serum treatmentsdTAG-13 (Tocris, catalog 660520) was added at a final concentration of 500 nM for the indicated time points in each experiment with a corresponding DMSO control. For all serum inductions, cells were first seeded in full serum media (DMEM-7% FBS-1% P/S). Upon reaching 80% confluence, cells were washed with 1× DPBS (ThermoFisher Scientific, catalog 14190250), and then serum-free (DMEM-1% P/S) media was added to cells. DMSO/dTAG was added 40 h later followed by the addition of 18% serum-containing media (DMEM-18% FBS-1%P/S) for the time points indicated in each experiment.Cloning of dTAG constructsFor cloning of dTAG constructs as well as making the HCT116 KAP1dTAG cell line, published procedures were followed with minor modifications90. Briefly, the gRNA was designed from a combination of Benchling for off-target scores and the Washington University gRNA designer tool for structural scores (https://crisprdb.org/wu-crispr-website/index.html). The primers for both gRNA and arm cloning were designed based on this gRNA selection following the tutorial90. To clone the gRNA plasmid, custom-made DNA oligonucleotides (MilliporeSigma) were annealed with T4 ligase (NEB, catalog M0202S) and phosphorylated with T4 polynucleotide kinase (NEB, catalog M0201L) by incubating at 37 °C for 30 min, 95 °C for 5 min, and a ramp down to 25 °C at 5 °C/min. To prepare the sgRNA backbone vector, the empty universal cutting vector (pX330A-sgX-sgPITCh) was digested with BbsI-HF (NEB, catalog R0539S) for 1 h at 37 °C followed by a 15 min treatment with Quick CIP (NEB, catalog M0525S) and subsequent gel purification (QIAGEN, catalog 28606). Prepared oligonucleotides were then ligated into the digested backbone vector with T4 ligase at room temperature for 1 h followed by heat-inactivation and transformation into home-made DH5α competent cells followed by plasmid preparation and sequence verification using the primers listed in Supplementary Table 2. To clone arm plasmids, the pCRIS-PITChv2-dTAG-Puro, and pCRIS-PITChv2-dTAG-BSD plasmids were digested with MluI-HF (NEB, R3198S) followed by gel purification to prepare the linearized vector. To prepare the arm cassette, the original arm plasmid was utilized as a template for PCR using the designed arm primers to insert the KAP1 C-terminal homology arms using Q5® Hot Start High-Fidelity DNA Polymerase (NEB, catalog M0493L). The ~1-kb product was purified and Gibson assembly was performed (50 °C, 30 min) using 1.5 µL of NEBuilder HiFi Master Mix (NEB, catalog E2621S) and the digested vector and PCR product. The Gibson product was diluted and transformed into homemade DH5α competent cells, and plasmid DNAs were prepared and verified by Sanger sequencing. All plasmids and primers used in this study are listed in Supplementary Tables 1 and 2, respectively.Creation of HCT116 KAP1dTAG cellsTo generate HCT116 KAP1dTAG cells, we used a double selection approach in which both puromycin- and blasticidin-resistant dTAG cassette plasmids were transfected in tandem with a sgRNA plasmid. We selected cells with both antibiotics to target the two KAP1/TRIM28 alleles and single-sorted for expansion followed by western blot to ensure no presence of the endogenous (lower molecular weight) KAP1 protein band. Briefly, HCT116 parental cells were seeded in 6-well plates (~500,000 cells/well) and transfected ~24 h post-seeding with three plasmids (BSD arm plasmid, Puro arm plasmid, and gRNA plasmid) targeting the C-terminus of KAP1 at a ratio of 1:1:1 (0.33 µg each). Before transfection, the media was changed into 1 mL of fresh DMEM. Two mixes were prepared: (1) a DNA mix consisting of 1 µg of total plasmid DNA (0.33 µg of each plasmid) into 100 µL of OPTIMEM (Gibco, catalog 11058021) and a Lipofectamine 2000 (ThermoFisher Scientific, catalog 52887) mix of 3 µL incubated in final 100 µL OPTIMEM. The Lipofectamine 2000 mix was added dropwise to the DNA mix and incubated for 10 min at room temperature. After incubation, the 200 µL mix was added dropwise to cells, and the media was changed into 2 mL of DMEM media after 5 h. Two days post-transfection, selection media (1.5 µg/mL puromycin (ThermoFisher Scientific, catalog 227420500) and 10 µg/mL blasticidin (ThermoFisher Scientific, catalog BP264750) in complete DMEM) was added to the cells for 14 days to induce targeting of both KAP1/TRIM28 alleles, at which point stable colonies began to expand. A single-selection approach including 0.5 µg of only PURO arm plasmid and 0.5 µg of gRNA plasmid was included as control. Cells were re-split in a 6-well plate, grown for 4 days, and then expanded to a 10-cm tissue culture dish. Population lysates were collected and subjected to KAP1 western blot to verify targeting efficiency. Cells were single-cell sorted in 96-well plates at the UTSW Children’s Research Institute Moody Foundation Flow Cytometry Core in complete DMEM (200 µL per well with no antibiotic). Cells grew in single-cell format for ~4–5 weeks prior to slowly expanding to 10-cm tissue culture dishes (24-well first, 6-well second, and then finally to 10-cm dishes). To verify KAP1 targeting, anti-KAP1 western blots were performed.Western BlotsAll western blots were run on 10–12% SDS-PAGE gels and transferred on nitrocellulose membranes (Bio-Rad, catalog 1620115) using the Bio-Rad Trans-Blot Turbo Transfer System, blocked for 1 h in 5% Milk (or BSA) + Tris-buffered saline-Tween-20 (TBST), probed with primary antibody (see Supplementary Table 3) in 5% Milk (or BSA) + TBST. Primary and secondary antibody concentration and time of incubation are indicated in Supplementary Table 3. Blots were exposed using either Clarity Western ECL (Bio-Rad, catalog 1705060) or SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, catalog 34095) or utilizing the starbright secondary channel.Cell confluence assayFor the cell growth assay, cells were treated with either 8 h DMSO or dTAG and then the IncuCyte S3 Live Cell Imaging System (Essen Bioscience) was used for cell proliferation assays. 2000 cells/well were seeded into 96-well plates and imaged every 6 h over 144 h (6 days). Phase contrast images were used to calculate cell confluence using the IncuCyte software and the cell confluence at each time point was normalized to time 0.RNA extraction and RT-qPCRFor RT-qPCR, cells were treated as described and RNA was extracted using the Zymo Quick-RNA MiniPrep Kit (Zymo Research, catalog R1055) following the kit instructions. RNA was quantified using the DeNovix DS-II FX+ Spectrophotometer and all RNAs were diluted to the same yield and re-quantified prior to cDNA synthesis reaction. To prepare cDNA, 2 μg of RNA was incubated with 1/200th of a unit of hexanucleotide primers (MilliporeSigma, catalog H0268-1UN) and 1 μL of 10 mM dNTP mix (NEB, catalog N0447L) for 5 min at 70 °C. Next, 2 μL of 10× M-MuLV Reverse Transcriptase buffer and 1 μL of M-MuLV Reverse Transcriptase (NEB, catalog M0253L) were added to each sample (final volume 20 μL) and incubated at 42 °C for 1 h. The reaction was inactivated at 70 °C for 10 min and samples were diluted by adding 80 μL with H2O. For qPCR, 1 μL of diluted cDNA, 5 μL PowerUp™ SYBR™ Green Master Mix for qPCR (ThermoFisher Scientific, catalog A25741), 3 μL of H2O, and 1 μL of 5 μM primer mix was used for each well in a 96-well plate. All primers used for RT-qPCR analysis are listed in Supplementary Table 2. Samples were amplified for 40 cycles using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System. All RT-qPCR data was analyzed using the ΔΔCt method where each RNA for each sample was normalized to the DMSO-Serum 0 min (S0) sample and then each target gene was normalized to two control genes (RPL19 and GAPDH) using the geometric mean of their expression.RNA-SeqRNAs for RNA-Seq were prepared as described for RT-qPCR. For RNA-Seq library preparation, 1 μg of RNA was used as input with 2 μL of a 1:100 dilution of ERCC RNA Spike-In Mix (ThermoFisher Scientific, catalog 4456740) and the KAPA RNA Hyper+RiboErase HMR kit was used (Roche, catalog 8098131702) following the manufacturer’s instructions for library preparation using KAPA beads (Roche, catalog KK8001). The following steps were performed: Oligo hybridization and rRNA depletion, KAPA bead cleanup, digestion with DNAse, and another KAPA bead cleanup as described in the kit protocol. RNA elution, fragmentation (8 min at 94 °C), and priming were performed followed by first-strand synthesis and second-strand synthesis/A-tailing. Adapter ligation (15 min at 20 °C) was performed with 7 μM NEBNext® Adaptor for Illumina® (NEB, kit catalog E6446L) followed by a 3 μL USER enzyme digest (15 min at 37 °C). This was immediately followed by two cleanups (first with 0.63× KAPA beads and second with 0.7× PEG/NaCl solution). After adapter ligation, samples were PCR amplified using Illumina indexed primers (NEB, E7335L) for 8 cycles. Following a final KAPA bead purification and elution with 20 μL of 10 mM Tris-HCl pH = 8.0, size distribution and quality of libraries was determined using the Agilent Tapestation DNA ScreenTape (Agilent, catalog 5067-5582). Libraries were then quantified using the Qubit dsDNA HS Assay (ThermoFisher Scientific, catalog Q32851) and sequenced at ~33E6 paired-end reads/sample with 50-bp length using a NextSeq 500 (Illumina) at the UT Southwestern McDermott Center Sequencing Core. Three biological replicates per treatment condition were submitted.TT-SeqFor TT-Seq, the protocol from Patrick Cramer’s lab was followed with minor modifications91. Three 10-cm tissue culture dishes per condition were seeded such that cells reached 75% confluence 3 days post-seeding. On the third day, cells were treated with DMSO or dTAG for 8 h, after which 500 μM 4-Thiouridine (MilliporeSigma, catalog T4509) was added to the media for 10 min. An extra dish was seeded per condition and ~12E6 cells were counted per plate and thus 36E6 cells/sample were used to prepare RNA. Cells were washed with 5 mL of 1× PBS once and then immediately 1 mL of TRIzol™ Reagent (ThermoFisher Scientific, catalog 15596026) was added and followed by 10 min of rocking. Cells were scraped off the plate, transferred to a 15 mL tube, and then homogenized by pipetting ~10 times. Cells were placed at –80 °C until ready for RNA extraction. To isolate total RNA, 200 μL chloroform was added to the 1 mL of sample (3 tubes per replicate) and vortexed 15 s followed by centrifugation (13,000 × g, 15 min, 4 °C). The upper, aqueous layer (~490 uL) was added to a fresh tube with 1 μL Glycoblue and an equal volume of Isopropanol (~490 μL) followed by mixing, incubation at room temperature for 10 min, and then centrifugation (13,000 × g, 10 min, 4 °C). The pellet was washed twice with 1 mL 75% Ethanol followed by centrifugation (7500 × g, 5 min, 4 °C). After drying, the pellet was resuspended in 320 μL RNAse-free water per replicate, quantified by nanodrop, and diluted to 750 ng/μL followed by sonication using a Q800R3 Qsonica (50% amplitude with 3 cycles 30 s ON, 30 s OFF at 4 °C). The RNA was denatured at 65 °C for 10 min, placed on ice for 5 min, and diluted to 150 μg/700 μL to prepare for biotinylation. 10× Biotinylation buffer (100 mM Tris-HCl pH = 7.5, 10 mM EDTA pH = 8.0) was prepared along with 5× (1 mg/mL) EZ-Link™ HPDP-Biotin (MilliporeSigma, catalog 21341) in N,N-Dimethylformamide (Acros Organics, catalog 279600010). 300 μgs total RNA was used per replicate, and thus each condition had 2 × 1000 μL biotinylation reactions. A mix of Biotinylation Buffer (100 μL per reaction) and Biotin-HPDP was made (200 μL per reaction) and added to 700 μL of diluted RNA, the samples were mixed and then nutated for 2 h at room temperature while covered in foil. To purify RNA after biotinylation, 500 μL of each reaction was transferred to a new tube followed by the addition of an equal volume of chloroform (500 μL). Samples were vortexed, incubated for 3 min, and then centrifuged (1500 × g, 5 min, 4 °C), followed by transfer of the aqueous layer (~300 μL) to 2 new tubes per sample (~600 μL each) and then precipitation with 1/10th volume of 5 M NaCl (~60 μL), 1 μL glycoblue, and 1× isopropanol (~600 μL). Samples were vortexed, centrifuged (13,000 × g, 30 min, 4 °C), washed two times with 75% ethanol, and then resuspended in 500 μL of RPB buffer (10 mM Tris-HCl pH = 7.5, 1 mM EDTA pH = 8.0, 300 mM NaCl). To isolate biotinylated RNA, 200 uL of Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, catalog 65601) was aliquoted per sample and washed 4 times with 1 mL beads wash buffer (10 mM Tris-HCl pH = 7.5, 1 mM EDTA pH = 8.0, 50 mM NaCl) and finally suspended in 1 mL beads wash buffer per sample plus 0.1% polyvinylpyrrolidone (ThermoFisher Scientific, catalog BP431-100). Beads were then nutated at room temperature for 10 min, washed one time with 1 mL beads wash buffer, and then suspended in RPB (200 μL per reaction). The biotinylated RNA product was denatured at 65 °C for 5 min, placed on ice for 2 min, and then 200 µL of blocked beads to each sample was added followed by a 30 min incubation with rotation at room temperature. After 30 min, beads were washed 5 times with 4sU wash buffer (10 mM Tris-HCl pH = 7.5, 1 mM EDTA pH = 8.0, 1 M NaCl, 0.1% Tween-20). Biotinylated, 4sU-RNA was eluted from beads twice by adding 75 μL of 0.1 M DTT to beads and nutating for 15 min at room temperature, yielding a final elution volume of ~150 μL. 4sU-RNA was purified and concentrated using the Zymo RNA Clean and Concentrator Kit (Zymo Research, catalog R1013) following kit instructions and RNA was quantified using the Qubit™ RNA High Sensitivity (HS) Assay (ThermoFisher Scientific, Q32852). Libraries were prepared similarly to RNA-Seq except with the minor modifications described here. First, 2 μL of a 1:4000 ERCC Spike-in mix was added to approximately ~50 ngs of nascent RNA as input to library preparation. Fragmentation was done for 6 min at 94 °C and 13 cycles of PCR were used for PCR amplification step. Libraries were quality-checked with Tapestation and Qubit and then sequenced at ~100E6 paired-end reads/sample with 50-bp length using a NextSeq 500 (Illumina) at the UT Southwestern McDermott Center Sequencing Core. Two biological replicates per treatment condition were submitted.ChIP-SeqAll ChIP-Seq experiments were performed as previously described45 with minor modifications. For each ChIP, a 1 × 15-cm tissue culture dish (yielding ~30E6 cells/plate) was utilized and seeded according to the experimental design in each figure (DMSO/dTAG ± serum at different time points), and 20E6 cells were utilized per ChIP. For all Pol II ChIPs (RPB3, Ser5P Pol II, Ser2P Pol II), cells were crosslinked with 0.5% methanol-free formaldehyde (ThermoFisher Scientific, 28908) by adding directly to the tissue culture dish in media at room temperature for 10 min with rocking and neutralization with 150 mM glycine for 5 min with rocking. For all other ChIP-Seq experiments (HA (KAP1), SPT5, pSPT5, CDK9, CDK7, TFIIB, MED1), 1% formaldehyde for 10 min was used. For all experiments, the media was removed and cells were washed twice with cold 1× PBS, cold PBS was then added to the plate and then cells were collected by scraping. Cells were centrifuged (1000 × g, 5 min, 4 °C), and pelleted, flash frozen in liquid nitrogen, and frozen at –80 °C until ready to use. To perform the ChIP, cells were resuspended in 4 mL/dish of Farnham Lysis Buffer (5 mM PIPES pH = 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, 1× Protease Inhibitor (RPI, catalog P50900-1)), counted by hemocytometer, resuspended to 10E6 cells/mL, nutated for 30 min at 4 °C, and then centrifuged to isolate nuclei (1000 × g, 5 min, 4 °C). The supernatant was removed and nuclei resuspended in Szak’s RIPA Buffer (50 mM Tris-HCl pH = 8.0, 1% NP-40, 150 mM NaCl, 0.5% Na-Deoxycholate, 0.1% SDS, 2.5 mM EDTA pH = 8.0, 1 mM PMSF, and 1× Protease Inhibitor) at a concentration of 25E6 nuclei/mL. The chromatin was sheared using a Q800R3 Qsonica (50% amplitude with 25 cycles 30 s ON, 30 s OFF at 4 °C) to a DNA molecular weight range of 200–400-bp. After sonication, chromatin was centrifuged (21,000 × g, 15 min, 4 °C) and the supernatant was taken as clarified chromatin. For ChIP-Seq experiments that contained Drosophila spike-in, 50 ng of spike-in chromatin (Active Motif, catalog 53083) was added to each chromatin sample. Sheared chromatin was pre-cleared by incubating with 25 μL of Szak’s RIPA equilibrated Protein G Dynabeads (ThermoFisher Scientific, 10003D) for 1 h at 4 °C. To equilibrate beads for pre-clearing, a master mix of beads was incubated with RIPA buffer, nutated for 5 min, and placed on a magnet to remove supernatant three times. To bind antibody to beads, 100 μL of Protein G Dynabeads per sample were equilibrated with 1× PBS + 0.05% Tween-20 and resuspended to a final volume of 250 μL per ChIP. The corresponding antibody (see Supplementary Table 3 for antibody details) was then added to the 250 µL beads, and the bead-antibody mix was nutated for 1 h at 4 °C. For samples that contained Drosophila spike-in, 2.5 μg of antibody specific for Drosophila H2Av (Active Motif, catalog 61686) was added to each tube. Antibody bound beads were then blocked in Szak’s RIPA Buffer + 5% BSA for 1 h at 4 °C with rotation. Pre-cleared sheared chromatin was then added to beads and incubated overnight at 4 °C with rotation. Beads from each sample were washed 2 times with 900 μL of Szak’s RIPA Buffer, Low-Salt Buffer (0.1% SDS, 1% NP-40, 2 mM EDTA pH = 8.0, 20 mM Tris-HCl pH = 8.0, 150 mM NaCl), High-Salt Buffer (0.1% SDS, 1% NP-40, 2 mM EDTA pH = 8.0, 20 mM Tris-HCl pH = 8.0, 500 mM NaCl), LiCl buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA pH = 8.0, 20 mM Tris-HCl pH = 8.0), and TE Buffer (10 mM Tris-HCl pH = 8.0, 1 mM EDTA pH = 8.0). After the final wash, samples were pulse spun in a table-top centrifuge to get rid of residual buffer before placing it on the magnet. Samples were then eluted from beads in 100 μL of elution buffer (100 mM NaHCO3 pH = 8.0, 1% SDS) for 30 min at 65 °C while vortexing every ~10 min. Input samples (40 μL) were volumed up to 100 μL by adding 60 μL elution buffer. DNA was eluted by placing the beads on a magnet, elutions transferred to new tubes, and de-crosslinked for 4 h at 65 °C with 100 μL volume of de-crosslinking buffer (500 mM NaCl, 2 mM EDTA pH = 8.0, 20 mM Tris-HCl pH = 6.8, 0.5 mg/mL Proteinase K (Epicentre, catalog MPR-90938)). ChIP DNA was purified and concentrated with the Zymo ChIP DNA Clean & Concentrator (Zymo Research, catalog D5201). ChIP samples were quantified by Qubit, and libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, catalog KK8502) following the manufacturer’s instructions. Briefly, samples underwent End Repair & A-tailing followed by adapter ligation with 300 nM to 1.5 µM NEB adapter depending on initial yield. Following a post-ligation cleanup, PCR amplification was performed with anywhere between 8-14 total cycles depending on initial yield. A KAPA bead cleanup was performed (1×) followed by size selection. For size selection, the first cleanup utilized 35 μL KAPA beads where the final supernatant contains the DNA of interest. The second and final cleanup utilized 10 μL of beads where the beads contain the bound, desired DNA. Following elution with 20 μL of 10 mM Tris-HCl pH = 8.0, libraries were quality-checked with Tapestation and Qubit, and then sequenced at ~33E6 paired-end reads/sample with 50-bp length using a NextSeq 500 (Illumina) at the UT Southwestern McDermott Center Sequencing Core. Two biological replicates of each ChIP-Seq for HA, Pol II, and Ser2P Pol II were completed, while one replicate of SPT5, pSPT5, CDK9, CDK7, TFIIB, MED1, and Ser5P Pol II were performed in each condition.PRO-SeqFor PRO-Seq experiments, we followed the qPRO-Seq protocol92 with minor modifications. Cells were seeded in 10-cm tissue culture dishes and treated as indicated and were ~90% confluent prior to collection. For each PRO-Seq, 4E6 cells were utilized. To prepare permeabilized cells for run-on, cells were first washed twice on the plate with 5 mL of ice-cold 1× PBS. Then, 2.5 mL of ice-cold Cell Permeabilization Buffer (CPB) (10 mM Tris-HCl pH = 8.0, 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.1% NP-40, 0.5 mM DTT, 0.05% Tween-20, 0.1% Triton X-100, 10% Glycerol, 1× protease inhibitor, and 2 µL SUPERase-In RNase inhibitor (ThermoFisher Scientific, catalog AM2696) per 10 mL) was immediately added to the tissue culture dish. Triton X-100 was added to CPB to increase permeabilization. The tissue culture dishes were placed on ice and then immediately scraped with a cell scraper. At this point, cells were collected in a 15 mL tube, placed on ice for 5 min, checked for permeabilization by trypan blue staining (>95%), and then centrifuged in a swinging bucket rotor (1000 × g, 4 min, 4 °C). Following this, cells were handled using cut tips. The supernatant was removed and samples were resuspended in 1 mL of Cell Wash Buffer (CWB) (10 mM Tris-HCl pH = 8.0, 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.5 mM DTT, 10% Glycerol, 1× protease inhibitor, and 2 µL SUPERase-In RNase inhibitor per 10 mL) and centrifuged (1000 × g, 4 min, 4 °C) for a total of 2 washes. After the second wash, samples were resuspended in 1 mL total Cell Freeze Buffer (CFB) (50 mM Tris-HCl pH = 8.0, 5 mM MgCl2, 0.5 mM DTT, 40% Glycerol, 1.1 mM EDTA pH = 8.0, and 2 µL SUPERase-In RNase inhibitor per 10 mL), counted by hemocytometer, and then spun down in 1.5 mL tubes in an angled rotor centrifuge (1000 × g, 5 min, 4 °C). Samples were resuspended in 52 μL of CFB for every 4E6 cells, flash frozen in liquid nitrogen, and then frozen at –80 °C until ready to use. For run-on assays, two biotinylated NTP’s (UTP and CTP, PerkinElmer, catalog NEL543001EA and NEL542001EA) were used with unbiotinylated ATP and GTP (MilliporeSigma, catalog 11277057001) and 2× ROMM buffer (10 mM Tris-HCl pH = 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 40 µM Biotin-11-CTP, 40 µM Biotin-11-UTP, 40 µM ATP, 40 µM GTP, 1% Sarkosyl (MilliporeSigma, catalog L5125) and 1 μL SUPERase-In RNase inhibitor per reaction) was prepared exactly as recommended. 50 μL of pre-heated 2× ROMM buffer was added to 50 μL of cell suspension, pipetted with a cut 200 μL tip 20–25 times quickly, and incubated (with 700 RPM shaking) for 5 min, after which 250 μL of Trizol LS (ThermoFisher Scientific, catalog 10296028) was immediately added, mixed by pipetting, vortexed, and placed on ice. 65 µL chloroform was added to each sample, vortexed, incubated on ice for 3 min, and centrifuged (20,000 × g, 8 min, 4 °C). Approximately ~150 μL of the aqueous phase was collected in a new tube followed by addition of 1 μL Glycoblue (ThermoFisher Scientific, catalog AM9515) and 2.5× volumes of 100% ethanol, and samples were then vortexed for 5 s and centrifuged (20,000 × g, 20 min, 4 °C). The supernatant was removed, washed with 75% ethanol once with gentle inversion and pulse spun. RNA pellets were airdried followed by resuspension in 30 μL of RNAse-free water. RNA was denatured for 30 s at 65 °C, snap-cooled on ice, and then fragmented with 7.5 μL of cold 1 M NaOH on ice for 10 min. 75 μL of 0.5 M Tris-HCl pH = 6.8 was added and mixed by pipetting, and then samples were passed through a calibrated Micro Bio-Spin™ P-30 Gel Columns, Tris Buffer (RNase-free) (Bio-Rad, catalog 7326250) following the manufacturer’s instructions. The volume of each sample was brought up to 200 μL with water, and 1 uL glycoblue, 8 μL NaCl, and 500 μL 100% ethanol were added, vortexed, and then centrifuged (20,000 × g, 20 min, 4 °C). Ethanol was removed and RNA pellets were stored in –80 °C overnight. The next day, 75% ethanol was used to wash the pellet, spun down, and then pellets were resuspended in 6 μL of RNAse-free water. 1 μL of 10 μM VRA3 oligo (see Supplementary Table 2) was added to the RNA and samples were denatured for 30 s at 65 °C and snap-cooled on ice. A buffer containing T4 RNA Ligase 1 (ssRNA ligase) (NEB, catalog M0204L) was added to samples for ligation for 1 h at 25 °C. 10 µL/sample of Dynabeads™ MyOne™ Streptavidin C1 Beads (ThermoFisher Scientific, catalog 65001) were then equilibrated by removing storage buffer by placing on a magnet, washed once in 1 mL bead preparation buffer (0.1 M NaOH and 50 mM NaCl) and twice with 1 mL binding buffer (10 mM Tris-HCl pH = 7.5, 300 mM NaCl, 0.1% Triton X-100, 1 mM EDTA pH = 8.0, and 2 μL SUPERase-In RNase inhibitor per 10 mL). Each wash was done by adding the indicated buffer, flipping the tube on the magnet twice, and then removing the buffer without letting the beads dry. Beads were resuspended in 25 µL binding buffer per sample and placed on ice until ready to use. After 3′ adapter ligation, 55 μL of binding buffer and then 25 μL of beads were added to each sample and rocked for 20 min at room temperature to bind biotinylated, nascent RNA to the beads. Beads were washed with 500 μL High-Salt Buffer (HSB, 50 mM Tris-HCl pH = 7.5, 0.5% Triton X-100, 2 M NaCl, 1 mM EDTA pH = 8.0 and 2 μL SUPERase-In RNase inhibitor per 10 mL), then 500 μL Low-Salt Buffer (LSB, 5 mM Tris-HCl pH = 7.5, 0.1% (v/v) Triton X-100, 1 mM EDTA pH = 8.0 and 2 μL SUPERase-In RNase inhibitor per 10 mL), and then resuspended in a buffer containing T4 polynucleotide kinase (NEB, catalog M0201L) and incubated for 30 min at 37 °C. Samples then underwent 5′ DeCapping using a buffer containing RppH enzyme (NEB, catalog M0356S) for 1 h at 37 °C, which was followed by 5′ adapter ligation on-beads (VRA5 oligo (see Supplementary Table 2) was added to each sample and then RNA-bound beads were denatured as described for 3′ adapter ligation) for 1 h at 25 °C. After 5′ ligation, the beads were washed with both HSB and LSB, resuspended in 300 μL of Trizol, vortexed, and incubated on ice for 3 min. 60 μL chloroform was added to each sample, vortexed, incubated on ice for 3 min, and then centrifuged (20,000 × g, 8 min, 4 °C). Approximately ~180 μL of the aqueous phase was collected in a new tube followed by an addition of 1 μL Glycoblue and 2.5× volumes of 100% ethanol. Samples were then vortexed and centrifuged (20,000 × g, 20 min, 4 °C). The supernatant was removed, washed with 75% ethanol once with gentle inversion and pulse spin, and RNA pellets were airdried followed by resuspension in 13.5 µL Reverse Transcriptase (RT) resuspension mix to begin cDNA synthesis, which consisted of 4 μL of 10 μM RPI oligo (see Supplementary Table 2) and dNTPs. Samples were denatured at 65 °C for 5 min, placed on ice, and resuspended in an RT master mix consisting of Maxima H Minus RT enzyme (ThermoFisher Scientific, catalog EP0752), and cDNA synthesis was performed on a thermal cycler (50 °C for 30 min, 65 °C for 15 min, and 85 °C for 5 min). 2.5 μL of a 10 μM RPI-X (designated RPI indexing primer) was added to each cDNA synthesis reaction followed by 78.5 μL of PCR amplification master mix (consisting of Q5® High-Fidelity DNA Polymerase) and PCR was performed with a total of 14 cycles following the conditions (56 °C extension) in the original protocol. The samples were purified using 180 μL of KAPA pure beads, and eluted in 15 μL of 10 mM Tris-HCl pH = 8.0. Libraries were quality-checked with Tapestation and Qubit and then sequenced at ~66E6 paired-end reads/sample with 50-bp length using a NextSeq 500 (Illumina) at the UT Southwestern McDermott Center Sequencing Core. If samples had excess adapter dimer contamination, they were subsequently electrophoresed on a 2% agarose gel (80 V for 60 min) followed by gel excision in a cold room, purification by column (QIAGEN, catalog 28606), and additional TapeStation and Qubit analyses before sequencing. Two biological replicates per treatment condition were submitted.RNA-Seq and TT-Seq data analysisDetailed scripts for pre-processing and downstream analysis, tutorials, and files needed to run the analysis for this paper are under our lab GitLab page. Briefly, raw fastq data files were run through fastqc/0.11.8 and low-quality reads/adapter contaminations were removed using trimgalore/0.6.4. Reads were mapped to the hg38 human reference genome using star/2.7.3a. Spike-ins were mapped to a fasta file of ERCC sequences provided by the manufacturer. featureCounts (subread/1.6.3) was used to calculate counts across the entire gene of protein-coding genes using a .gtf file containing all protein-coding genes as well as ERCC spike-ins. EDASeq/2.32.0, RUVSeq/1.32.0, and EdgeR/3.40.1 commands were used for spike-in normalization and differential gene expression analysis of genes that contained at least 10 counts in 6 samples (RNA-Seq) and 50 counts in 4 samples (TT-Seq) was performed. Differential expression analysis files are provided as a table in Supplementary Data labeled ‘DEG Analysis File’. IEGs were identified by Log2FC values between DMSO-Serum Starved (SS) and DMSO-Serum (S). Log2FC values for each cluster are described by the following: 2-fold IEGs (Log2FC > or = 1.0, n = 236), 4-fold IEGs (Log2FC > or = 2.0, n = 69), 8-fold IEGs (Log2FC > or = 3.0, n = 34), 16-fold IEGs (Log2FC > or = 4.0, n = 16). non-DE genes (n = 10,361) were identified as genes that were less than 1.4-fold up or downregulated and were expressed by parameters described above. Volcano plots were made using EnhancedVolcano/1.16.0 from Bioconductor. Pathway analysis was performed using Enrichr93.ChIP-Seq analysisDetailed scripts, tutorials, and input files needed to run all analyses for ChIP-Seq analysis for this paper are under our lab GitLab page. Briefly, raw fastq data files were run through fastqc/0.11.8 and low-quality reads/adapter contaminations were removed using trimgalore/0.6.4. Reads were mapped to the hg38 human reference genome using bowtie2/2.4.2. Reads were also mapped to the dm6 Drosophila genome to extract reads originating from the spike-in chromatin for ChIPs that were normalized using spike-ins. Duplicates were marked and removed using picard/2.10.3 and then files were sorted and indexed using samtools/1.6 in preparation for bigWig generation. Normalized bigWig’s were made from sorted bam files using the bamCoverage command using deepTools/2.3.594. The following ChIP-Seqs were normalized using Drosophila spike-ins: HA, SPT5, pSPT5, CDK9, CDK7, TFIIB, and MED1. The following ChIP-Seqs were normalized to read depth (counts per millions or CPM using deepTools): Pol II, Ser5P Pol II, Ser2P Pol II. For spike-in normalization, scaling factors (Supplementary Table 5) were defined using deepTools command multiBamSummary and then inputted into bamCoverage for bigWig generation for normalization. Importantly, all spike-in normalized datasets were compared to read-depth normalized datasets to ensure quality and agreement. Ser5P Pol II and Ser2P Pol II ChIP-Seq data were normalized to Pol II ChIP-Seq data using deepTools command bamCompare. Detailed alignment statistics for both human genome reads and spike-in coverage are included in Supplementary Table 5. The Integrated Genome Viewer (IGV) was used for visualization.PRO-Seq analysis and proxy rate analysisScripts for running PRO-Seq analysis and performing the Proxy Elongation Rate analysis using the ROCC method are under our lab GitLab page. The PRO-seq libraries were analyzed using the Proseq2.0 pipeline95. Briefly, raw fastq data files were run through fastqc/0.11.8 and low-quality reads/adapter contaminations were removed using Cutadapt/2.5. Reads were subsequently aligned to the human reference genome hg38 using BWA/0.7.5. The aligned bam files were converted into RPKM-normalized bigWig format using deepTools/2.3.594 and bedGraphToBigWig96 program to visualize in IGV. For the Proxy Rate analysis, a set of ROCC’s for each 50-bp bin in the set of IEGs is estimated for the 0, 5, and 10 min PRO-Seq data (DMSO/dTAG) utilizing bigWig files that contained the normalized read counts (coverage). ROCC benchmarking, performed as described for the serum dataset in this paper, was done using a previous estrogen-stimulated dataset in MCF7 cells23. A generalized linear model was applied to the overall intensities in the datasets to determine the slope of each condition at each 50-bp bin across the three time points (the ROCC value). Each 50-bp bin ROCC value for each gene is averaged to form a final Proxy Rate value. The generalized linear model was done by fixing the y-intercept to be 0 to decrease the influence of the serum 0 time point, which often contained low or no signal affecting the final rate calculations. To ensure that this did not drastically affect the analysis, a secondary Proxy Rate analysis was conducted by taking the normalized measure of the coverage at every 50-bp bin for the 0 min time point and subtracting this from both the 5 and 10 min points separately, and then dividing by 5 and 10, respectively. This analysis allows the serum 0 time point to exert maximal influence and yielded similar results. All analysis was completed using the R programming language, and custom scripts that utilized the ‘rtracklayer’ library97 for interacting with the bigWig files. Benchmark validation of the ROCC method was done by comparison to the leading edge methodology followed by three common procedures, including Pearson’s correlation (the standard measure assuming normality), Spearman’s rank correlation (a non-parametric procedure that rank transforms the data and then performs regression), and Kendalls Tau (a non-parametric method which calculates the number of pairs that are in concordance, that is both x increases and y increases).Graphing and statistical analysisAll bar graphs, boxplots (Tukey plots), violin plots, and XY plots were created using GraphPad Prism v10. Statistical tests (two-sided unpaired Student’s t-test and paired Wilcoxon Signed-Rank Test) defined in each figure legend were also performed in GraphPad Prism v10.Metagene analysis and quantificationsScripts for metagene analysis and quantifications along with tutorials and our custom BED files are provided on the D’Orso lab GitLab page. RNA-Seq data was used to define and cluster IEGs. Metagenes were created using deepTools commands computeMatrix and plotProfile. Quantitation of signal was computed using computeMatrix. Promoter Proximal (PP) is defined as −100/+500-bp of TSS, Gene Body (GB) is defined as +500-bp of TSS to the pA site and 3′ end is pA site to 3000-bp downstream of each gene. Gencode annotation of Transcript Start Site (TSS) and Transcript End Site (TES) regions was used for preliminary analysis. However, after assessment of gene browser tracks of Pol II ChIP-Seq data, a custom BED file was created that more accurately represented the position of TSS and TES based on the criteria below, which is thoroughly explained in our GitLab. For TES, many genes had a Gencode TES site designation, which was well before the putative pA site. Thus, we used NCBI Gene reference sequences to define pA sites with integration of Pol II ChIP-Seq and Ser2P Pol II ChIP-Seq data to define the pA site for analysis. Additionally, Gencode annotation revealed a few IEGs that had the incorrect start site usage (e.g. mostly due to alternative start sites not utilized in HCT116) and thus a TSS-Seq repository (https://dbtss.hgc.jp/#kero:chr2:96145427-96145452:-) was used to more closely define putative TSS sites. Given that this designation was primarily manual, Gencode TSS and TES were used for non-DE metagene profiles. For HA, Pol II, and Ser2P Pol II ChIP-Seq data, two biological replicates were analyzed independently until the bigWig stage and were merged after visual inspection of individual browser tracks in IGV and Pearson correlation analysis using deepTools (see Supplementary Table 4 for Pearson correlation coefficients). All PRO-Seq experiments contained two biological replicates that were analyzed independently until the bigWig stage and were merged after visual inspection and correlation analysis (see Supplementary Table 4 for Pearson correlation coefficients). One replicate of each antibody in each condition was performed for the following ChIP-Seqs: SPT5, pSPT5, CDK9, CDK7, TFIIB, MED1, and Ser5P Pol II.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
