title: GSM5468078: ChIP-442-IAA-CTCF-R1-T1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed in the plates with 1% formaldehyde. Each 5 million cells were resuspended in 1 mL of hypotonic lysis buffer (20mM TRIS HCl pH8.0, 85mM KCl, 0.5% IGEPAL, 1X HALT protease inhibitor) and incubated 15 min on ice. Cells were then spun down 5 min, 500g, 4C, supernatant was removed and 5 million cells were resuspended in 300 uL of sonication buffer (20mM TRIS HCl pH8.0, 0.2% SDS, 0.5% Sodium Deoxycholate, 1X HALT protease inhibitor). Cells were sonicated using a bioruptor pico, at 4C, following the manufacturer instructions and with the following settings: 30 s on, 30 s off, for 15 cycles. 300 uL of chromatin from 5M cells was diluted with 1 200 uL of dilution IP buffer (20 mM TRIS HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% TRITON, 1X HALT protease inhibitor) and specific antibody was added. For end repair, 35 μL of sample were transferred to a PCR tube, then 15 μL of the end-repair mix (1X NEB ligation buffer (NEB B0202S), 17.5 mM dNTP mix, 7.5 U T4 DNA polymerase (NEBM0203L), 25 U T4 polynucleotide kinase (NEB M0201S), 2.5 U Klenow polymerase Polymerase I (NEB M0210L)) were added. The reactions were then incubated at 37°C for 30 minutes, followed by incubation at 75°C for 20 minutes to inactivate Klenow polymerase. An ampure beads purification was then performed (1.6X) and DNA was eluted in 41 uL of water. For A-tailing, dATP was added to the 3' ends by adding 9 μL of A-tailing mix (5 μL NEB buffer 2.1, 1 μL of 1 mM dATP, 5 U Klenow exo (NEB M0212S)) to the 41 μL of DNA sample from the previous step. The reaction was incubated in a PCR machine at 37°C for 30 minutes, then at 65°C for 20 minutes. An ampure beads purification was then performed (1.6X) and DNA was eluted in 40 uL of water. The TrueSeq DNA LT kits Set A (Illumina, #15041757) was used for adapter ligation and PCR amplification. PCR primers were removed using Ampure XP beads. The ChIP-seq libraries were sequenced using 50 bp paired end reads on Illumina HiSeq4000.