title: GSM3671075: CTCF_HUDEP2_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: 10^7 expansion phase HUDEP-2 cells were collected and fixed with 1%   formaldehyde for 5 minutes at room temperature. Fixation was quenched   with 125 mM glycine. Cells were washed with ice cold PBS twice and   resuspended in 0.13 mL lysis buffer (50 mM Tris-HCl 8.0, 10 mM EDTA,   0.5% SDS) and sonicated in a microtube (Covaris, 520045) with Covaris   E220 ultrasonicator (Covaris). 0.12 mL sonicated chromatin was mixed   with 1 mL ChIP dilution buffer (20 mM Tris-HCl 8.0, 2 mM EDTA, 1% Triton   X-100, 300 mM NaCl, protease inhibitor), 20 mL Dynabeads protein G   (Thermo Fisher Scientific) and 3 mg antibody (CTCF, 07-729, Millipore).   After overnight rotating, the beads were washed with the following   buffers: twice with RIPA150, twice with RIPA500, 500 mM NaCl, 0.1%   Sodium Deoxycholate, 0.1% SDS), twice with LiCl buffer (10 mM Tris-HCl   8.0, 1 mM EDTA, 0.5% NP-40, 250 mM LiCl, 0.5% Sodium Deoxycholate),   twice with TE buffer (10 mM Tris-HCl 8.0, 1 mM EDTA). To elute and   decrosslink, 300 mL Elution buffer (50 mM Tris-HCl 8.0, 10 mM EDTA, 1%   SDS, 150 mM NaCl, 0.1 mg/mL Proteinase K) was added to the beads and   incubated at 65 degrees overnight. The eluted material was extracted   with phenol-chloroform, and DNA was precipitated by adding 750 mL   absolute ethanol. DNA was pelleted by centrifuging at 14000 rpm for 15   minutes, washed once with 75% ethanol, then dried and dissolved with 50   mL TE buffer. The quality of ChIP was verified by real-time PCR. To construct ChIP-seq library, we used NEBNext Ultra II DNA Library Prep   Kit (NEB) according to manufacturer   s protocol. Quality check of library   was carried out with Qubit and bioanalyzer. The library was sequenced in   the NextSeq 500 platform with NextSeq 500/550 High Output Kit v2 (75   cycles). Paired-end sequencing was performed (2x42 bp, 6 bp index).
attributes.tag: GEO Accession
attributes.value: GSM3671075