title: GSM3207214: Cdk12△_Clone36_plusDox_3E8_rep1_ChIPseq; Mus musculus; ChIP-Seq
library_construction_protocol: RNA Sequencing: Total RNA was harvested using Trizol Reagent (Thermo Fischer) following the manufacturer's protocol and subsequently DNase treated with Turbo DNase (Thermo Fishcer) under standard reaction conditions. RNA quality was assessed by the Agilent 2100 BioAnalyzer and only samples with a RIN value >= 9 were used for library preparation. ChIP sequencing: We modified a high resolution, micrococcal nuclease (MNase) digestion-based ChIP methodology (Skene, P. J. & Henikoff, S. A simple method for generating high-resolution maps of genome-wide protein binding. eLife 4, 1–9 (2015).; Skene, P. J., Hernandez, A. E., Groudine, M. & Henikoff, S. The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1. eLife 3, 1–19 (2014).) Briefly, 48 hours before cells were harvested, 10e6 CDK12 cells were plated in 15 cm dishes either in +dox or –dox ES media. Cells were crosslinked directly in media on the plate in 1% methanol-free formaldehyde (Thermo Fisher) for 10 minutes at room temperature. Crosslinking was quenched with 250mM glycine. Cells were washed 3x in 10mL chilled PBS and harvested by scraping. Cells were pelleted, washed in 10mL chilled PBS, and pelleted again. PBS was aspirated off the cells and the pellets were flash frozen in liquid nitrogen and stored at -80 degrees C. Pellets were thawed on ice and resuspended in 0.9mL of ChIP Lysis Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCL pH7.5) supplemented with 1x cOmplete Protease Inhibitors (Roche) and 1x Halt Phosphatase Inhibitors (Thermo Fisher) and lysed for 10 minutes at room temperature. Pellets were diluted in 8.1mL of ChIP Dilution Buffer (1% TritonX-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl ph7.5) supplemented with 1x cOmplete Protease Inhibitors (Roche) and 1x Halt Phosphatase Inhibitors (Thermo Fisher) and 3mM CaCl2. Each tube was pre-warmed at 37 degrees C for 2 minutes. 12 Units of micrococcal nuclease (MNase, Sigma) were added per tube and samples were digested for 30 min at 37 degrees C with rotation. The digestion reaction was quenched with the addition of 180uL 500mM EDTA and 360uL 500mM EGTA per tube. Samples were sonicated in 15mL polystyrene tubes in a BioRupter (Diagenode) for 20 cycles on high (1 cycle = 30 seconds ON/30 seconds OFF). Samples were cleared by centrifugation (max speed, 4 degrees C, 10min). Soluble material was transferred to a new tube and each sample (one pellet) was split into four 1.8mL parts. Lysate was incubated overnight at 4 degrees C plus rotation with 10ug or 10uL of acites fluid of the following antibodies: Total RNAPII (8WG16 and Rpb3), RNAPII CTD Ser2p (H5 and 3E10), and RNAPII CTD Ser5p (H14 and 3E8). After the overnight incubation, the IPs were incubated for 2 hours at 4°C as follows: The 8WG16 and Rpb3 IPs were incubated with 100uL of Protein G Dynabeads (Thermo Fisher). The H5 and H14 IPs were incubated with 100uL of Protein G Dynabeads pre-conjugated overnight with 20ug goat anti-mouse IgM (Thermo Fisher 31172) in Bead Preparation Solution (9 ChIP Dilution Buffer: 1 ChIP Lysis Buffer). The 3E10 IP was incubated with 100uL of Protein G Dynabeads pre-conjugated with 20ug goat anti-rat IgG (Thermo Fisher 31226). IPs were washed as follows 2 x 2mL LB3 (20mM Tris-HCl pH7.5, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100); 1x 2mL LB3+ (20mM Tris-HCl pH7.5, 500mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100); 1x 2mL Lithium Chloride Buffer (10mM Tris-HCl, pH 7.5, 250mM LiCl, 1mM EDTA, 1% NP-40), and 1x 2mL TE+50mM NaCl (10mM Tris-HCl, pH 7.5, 1mM EDTA, 50mM NaCl). After washing, beads were resuspended in 200uL of Extraction Buffer (50 mM Tris pH 8, 10mM EDTA, 5mM EGTA, 1% SDS). For WCE control, 100uL of soluble input material was added to 100uL of Extraction buffer. Samples were incubated overnight at 65°C +1000rpm shaking to elute IP and reverse crosslinks. 200uL of eluted samples was cleared of beads and added to 200uL TE pH 8. Samples were digested with 0.2mg/mL RNaseA for 1 hour at 37°C. 7 uL of CaCl2 solution (10mM Tris-HCl, pH 8 and 300mM CaCl2) and 4uL of 20mg/mL proteinase K were added to each sample and incubated for 1 hour at 55°C. Samples were extracted 1 x phenol:chloroform followed by 1 x chloroform and precipitated overnight at -80 degrees C with a standard NaCl, ethanol, and glycogen DNA precipitation. Pellets were washed 2x 1mL 70% ethanol, dried, and resuspended in 70uL 0.1x TE pH 8. RNA Sequencing: Libraries were made from 1ug of total RNA input using the TruSeq Stranded mRNA Library Prep Kit (Illumina RS-122-2102) with multiplexing barcodes, following the standard protocol with the following specifications: (1) 5 min RNA fragmentation time, (2) Superscript III (Thermo Fisher) was used for reverse transcription, (3) 15 cycles of PCR were used during the library amplification step, and (4) AMPure beads (Beckman Coulter) were used to size select/purify the library post PCR amplification instead of gel size selection. ChIP Sequencing: All of the ChIP material (70 uL resuspended) or 200ng of input material (WCE) was used to prepare libraries. Sample DNA was end-repaired for 30 min at 20°C in a 100uL reaction: 1x T4 DNA Ligase Buffer (New England Biolabs, NEB), 0.4 mM dNTPs (NEB), 15 U of T4 DNA Polymerase (NEB), 5 U of Klenow enzyme (NEB), 50 U of T4 PNK (NEB). Samples were purified by Invitrogen Purelink Kit (Thermo Fisher) following standard conditions and eluted in 33uL. 32uL of purified product was A-tailed for 37°C for 30 min in a 50uL reaction with 1x NEB Buffer 2, 2 uM dATP (NEB), and 15 U of Klenow exo- (NEB). Samples were purified by Invitrogen Purelink Kit and eluted in 11uL. Illumina genomic adapters were ligated onto 10uL of the purified product for 15min at 20°C in the following 50uL reaction with 1x Quick Ligase Buffer (NEB), 400nM of Y-shaped Adapter Oligo, and 5uL of Quick Ligase. The reaction was cleaned up by a double size selection with AMPure beads as described by the manufacturer with the initial size selection using a 0.9x AMPure ratio and keeping the supernatant (to select against large products – mononucleosomes and larger) followed by a 1.8x AMPure selection keeping the bead bound material (to select against adapter dimers and free adapters). Note: When switching between first and second size selection, we used the following formula provided by the manufacturer to calculate the amount of additional beads to add: (second ratio-first ratio)*volume transferred. Illumina adapter oligos were added to the size selected product in a 50 uL PCR reaction with 200uM dNTPs (NEB), 1x High Fidelity Phusion Buffer (NEB), 1uL Phusion Polymerase (NEB), 0.5uM forward Illumina oligo adapter, and 0.5uM reverse Illumina oligo adapter with 16x cycles of standard Phusion PCR conditions (annealing: 65°C 30 sec and extension: 72°C 30 sec). PCR products were AMPure purified (1.8x ratio) according to manufacturer guidelines. Libraries were run on the BioAnalyzer. Libraries with adapter dimer contamination were repurified with extra AMPure selections until adapter dimer contamination disappeared.