title: ChIP-seq of Homo sapiens: atrial cardiomyocyte differentiation at D20 from WT WTC11 rep2 - H3K27ac ChIP-seq
design: Cells were grown in one 6-well dish per replicate, with all 6 wells pooled together after dissociation for the crosslinking step (approximately 20 million cells). Cells were dissociated in 0.25% Trypsin-EDTA for 10 mins, followed by quenching in 15%FBS in DPBS. Cells were lifted off the dish and resuspended to single cells by repeatedly using P1000 pipette and then transferred to a 15 ml tube and centrifuged at 800 rpm for 5 min at room temperature. Pellets were washed once in 10 ml DPBS plus 1x protease inhibitors (DBPS+PI) before resuspending entire cell pellet in exactly 10 ml DPBS+PI. 16% methanol-free formaldehyde was added to a final concentration of 1% and samples incubated on nutator at room temperature for 10 min. Samples were quenched with 2.5 M glycine to a final concentration of 0.125 M and incubated on nutator at room temperature for 5 min, followed by centrifugation at 500 x g for 5 min at 4C. Samples were washed once in ice cold DPBS+PI, before being transferred to 2 ml protein lo-bind tubes, centrifuged at 2500 x g at 4C. for 5 min and cell pellets were snap frozen in liquid nitrogen and stored at -80C. Cell lysis for 10 min at 4C (cell lysis buffer recipe: 20 mM Tris-HCl pH 8.0, 85 mM KCl, 0.5% IGEPAL CA-630 and 1x protease inhibitors in ddH2O). Nuclear lysis for 30 min on ice  (nuclear lysis buffer recipe: 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 1x protease inhibitors in ddH2O). Samples were transferred to 1 ml millitube (Covaris #520130) and sheared using a Covaris S2 sonicator for 15 min at 5% duty cycle, intensity 5, 200 cycles per burst in a water bath maintained at 4C. 48 g of chromatin was used per sample. After diluting in dilution buffer, each ChIP sample was incubated rotating at 4C overnight with 10 g rabbit anti-H3K27ac (Active Motif, #39133) and the following day incubated with 50 l prewashed Pierce protein A/G beads for 4 hours at 4C. Bead washes were performed by resuspending beads in 1 ml buffer using P1000 pipette, spinning briefly and collecting on magnet. Wash buffer 1 recipe (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS and 1% Triton X-100 in ddH2O), wash buffer 2 recipe (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS and 1% Triton X-100 in ddH2O) and wash buffer 3 (250 mM LiCl, 1% IGEPAL CA-630 [Sigma #I8896], 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl pH 8.0 in ddH2O) and elution buffer (1% SDS, 100 mM NaHCO3 in ddH2O). Following the bead washes, DNA was eluted in 50 l shaking at 37C, transferring to new tube and repeating bead elution to pool eluates to a total volume of 100 l. Both ChIP and input samples were incubated at 37C for 30 min at 37C with 1 l RNase A, followed addition of 12 l 5 M NaCl and reverse crosslinking with 1 l proteinase K at 65C overnight. DNA was purified using ChIP DNA Clean and Concentrator kit (Zymo Research #D5205). For Illumina sequencing library preparation, the entire ChIP sample or 2 l of each input was used. End repair was performed in a 100 l reaction with 15 U T4 DNA polymerase (New England Biolabs #M0203L), 5 U Klenow fragment DNA polymerase (New England Biolabs, #M0210L), 50 U T4 PNK (New England Biolabs #M0201L), 400 M dNTP (Promega #U1511) and 1x T4 DNA ligase buffer w 10 mM ATP (New England Biolabs #B0202S) in nuclease-free water for 30 min at room temperature, followed by 1.6x AmpureXP purification. Entire eluate was used for A-tailing in a 50 l reaction with 1 mM dATP (New England Biolabs #N0440S), 15 U Klenow 3>5 exonuclease (New England Biolabs #M0212L) and 1x NEB buffer 2 in nuclease-free water for 30 min at 37C, followed by 1.6x AMPureXP purification. Entire eluate was used for adapter ligation in a 50 l reaction with 6000 U T4 DNA ligase (New England Biolabs #M0202L), 20 nM annealed and uniquely indexed adapters and 1x T4 DNA ligase buffer with 10 mM ATP (New England Biolabs #B0202S) in nuclease-free water for 2 hours at room temperature, followed by 1x AMPureXP purification. Adapters were prepared by annealing following HPLC purified oligos: 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T and 5Phos-GATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG where * represents a phosphothiorate bond and NNNNNN is a Truseq index sequence.The entire eluate was used for PCR amplification in a 50 l reaction with 1x NEB Next High-Fidelity 2x PCR master mix (New England Biolabs #M0541L), 10 M primers (F: 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA, R: 5-CAAGCAGAAGACGGCATACGAGAT) in nuclease free water, using thermocycler settings 98C for 30 sec; 16 cycles of 98C for 10 sec, 58C for 40 sec and 72C for 30 sec; 72C for 5 min and followed by 0.8x AMPureXP purification. DNA was quantified using Qubit Fluorometer and dsDNA HS Assay Kit and quality assessed on Agilent Bioanalyzer. Input is also the input for WT RAD21 rep3.
library_name: d20_atrial_CM_H3K27ac_rep2