title: GSM4221657: BLaER cell line, B_CTCF_ChIPseq_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked for 10 min using 1% formaldehyde and quenched using a final concentration of 0.125M glycin. Cell pellets were lysed by incubating 10 min on ice with 5mM Pipes pH 8, 85 mM KCL, 0.5 % IGEPAL, 1X protease inhibitor (Roche®). After centrifugation, pellets were incubated in 1% SDS, 10 mM EDTA pH 8, 50 mM Tris-HCL pH 8.1 and 1X PIC for 10 min on ice. Chromatin was sheared on a Bioruptor360 pico sonicator (Diagenode) at 4oC for 14 cycles of 30 sec ON and 30 sec OFF. After sonication, the solution was left on ice for 1h to allow SDS precipitation and clarified by centrifugation at 16,000g at for 10 min at 4oC. Supernatant was transferred in a new tube, 10 % was saved as input and the rest was diluted to 1.2 mL with 1X cold IP buffer (Diagenode). 10μg of anti-CTCF (Milipore, 07-729) was added followed by overnight incubation at 4oC on a rotator. 42 μL of beads (Unblocked Protein A beads, kch-503-008, Diagenode) were used per IP after blocking them using 1% bovine serum albumin cold IP buffer for 15 min at 4oC under rotation. Blocked beads were added to the chromatin solution and incubated 3h at 4oC with rotation. Beads were then collected by centrifugation for 2 min at 3000 rpm at 4oC and washed 3 times with cold IP buffer and 2 times with cold TE buffer (10mM Tris pH 8, 1mM EDTA). Beads were then eluted with freshly prepared elution buffer (1% SDS, 0.1M NaHCO 3 ) and incubated 25 min at RT. The supernatant was transferred into a new tube and cross-linking was reversed by adding NaCl (final concentration 200mM) and incubating overnight at 65oC. Protein digestion was achieved by adding Tris pH 6.5 (40mM), EDTA pH 8 (10mM) and proteinase K (4μg/μL) and incubating 1h at 45oC. DNA was then purified by phenol chloroform isoamyl (25:24:1) extraction. The entire DNA sample was used to construct Illumina sequencing libraries. Library preparation was performed using the NEBNext DNA Library Prep Kit (New England BioLabs) with 2 μL NEBNext adaptor in the ligation step. Libraries were amplified for 14 cycles with Herculase II Fusion DNA Polymerase (Agilent) and were purified/size-selected with Agencourt AMPure XP beads (> 200 bp).
attributes.tag: GEO Accession
attributes.value: GSM4221657