GSM3671075: CTCF HUDEP2 ChIP; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
human umbilical cord blood-derived erythroid progenitor (HUDEP)
Sequenced DNA Library
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).