title: ChIP-seq of Homo sapiens: atrial cardiomyocyte differentiation at D20 from WTC11 with TBX5in/del rep2 - RAD21 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. 60 g of chromatin was used per sample. After diluting in dilution buffer, each ChIP sample was incubated rotating at 4C overnight with 9 g rabbit anti-RAD21 (Abcam, #ab992) 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.
library_name: TBX5_null_RAD21_rep2