GSM6506075: KELLY cells, dTAGv1 TADA2B, Input, 6 hours; Homo sapiens; ChIP-Seq
20 million cells were cross-linked with 1% formaldehyde in 22mL warm media for 10 minutes with rotation followed by quenching with the addition of 1.1mL of 2.5M glycine for 5 minutes. Cells were pelleted by centrifugation at 600xg and resuspended in 1mL cold PBS supplemented with protease inhibitors (Thermo Fisher cat.# 78430) and 1mM PMSF (Sigma Aldrich cat.# P7626). This wash was repeated twice for a total of three washes in PBS. Cells were resuspended in cytoplasmic and then nuclear lysis buffer, and cells were sheared on an E220 Covaris sonicator (Duty cycle: 5%; Peak power: 140W; Cycles per Burst:200; Temp: 4C; time 15 min). Sheared lysates were cleared by centrifugation at 15,000g for 10 minutes at 4C and incubated overnight with rotation at 4C with magnetic beads prebound with antibody, spike-in antibody (Active Motif cat.#61686), and spike-in chromatin (Active Motif cat.#53083). Following overnight incubation, beads were washed twice with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tri-HCl pH8.1, 150mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tri-HCl pH8.1, 500mM NaCl), twice with LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA 630, 1% deoxycholic acid, 10mM Tri-HCl ph8.1, 1mM EDTA), and once with TE. DNA was eluted in elution buffer (1% SDS, 0.1M NaHCO3). Cross linking was reversed overnight at 65C with 0.9% RNAse A, 0.9% Proteinase K, and 0.182M NaCl. DNA was isolated by adding Agencourt AMPure XP beads (Beckman Coulter cat.# A63880) at 1.2x volume, incubating 20 minutes, washing twice with 80% ethanol, and eluting into 10-20ul TE. ChIP-seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Subsequently, libraries were paired end sequenced on Illumina NovaSeq 6000.