title: ChIP-seq of Homo sapiens: atrial cardiomyocyte differentiation from WTC11 at D20 rep5
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 rubes, centrifuged at 2500 x g at 4C. for 5 min and cell pellets were snap frozen in liquid nitrogen and stored at -80C.  TBX5-bio cross-linked cells were thawed on ice and resuspended in 1 ml of ice cold cell lysis buffer (25 mM Tris-HCl, pH 7.4, 85 mM KCl, 0.25% Triton X-100 and 1x Protease inhibitors in ddH2O) and incubated at 4C rotating for 30 minutes. Sample were centrifuged at 2500 x g for 5 min at 4C and resuspended in 1 ml nuclear lysis buffer (0.5% 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. After shearing, 5 l of each sample was kept for reverse crosslinking and the remaining sonicated sample was transferred to a new protein lo-bind tube and snap frozen in liquid nitrogen. The 5 l sample was made up to 100 l and incubated 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. The following day the reverse crosslinked samples were purified using Qiagen MinElute PCR Purification kit (Qiagen #28006) and DNA quantified using Qubit Fluorometer and dsDNA HS Assay Kit (ThermoFisher Scientific #Q32854). Used an Agilent 2100 Bioanalyzer to verify that chromatin was sheared to fragments between 100-500bp in size. Based on these measurements, the amount of DNA in each lysed sample could be extrapolated and equal amounts of sheared chromatin could be used per ChIP. Sheared samples were thawed quickly at 37C and the equivalent of 55 g of chromatin was transferred to a 5 ml protein lo-bind tube for ChIP and made up to 1 ml in nuclear lysis buffer. 50 l (5%) of each sample was collected as input and stored at 4C until reverse crosslinking step. ChIP samples were then diluted 1:5 with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 167 mM NaCl and 1x protease inhibitors in ddH2O). 50 l of pre-washed Dynabeads MyOne Streptavidin T1 beads were added to each sample and incubated overnight with rotation at 4C. The following day, beads were collected using a magnetic stand and transferred to 2 ml protein lo-bind tubes and washed with 1 ml twice of the following wash buffers: wash buffer 1 (2% SDS in ddH2O), wash buffer 2 (0.1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 500 mM NaCl in ddH2O), wash buffer 3 (250 mM LiCl, 0.5% IGEPAL CA-630 [Sigma #I8896], 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl pH 8.0 in ddH2O) and TE buffer (10 mM Tris-HCl pH 7.5 and 1 mM EDTA ddH2O).  After adding each wash buffer, beads were vortexed for 15 s, spun briefly and collected against a magnet. DNA was eluted with 150 l elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0 in ddH2O) shaking at 1000 rpm at 65C overnight. The following day, beads were collected on a magnetic stand and supernatant was transferred to a new DNA lo-bind tube. Both ChIP and input samples were incubated at 37C for 30 min at 37C with 1.5 l RNase A, followed addition of 18 l 5 M NaCl and reverse crosslinking with 1.5 l proteinase K at 65C for 6 hours. DNA was purified using Qiagen MinElute PCR Purification kit (Qiagen #28006). 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.