GSM5463913: CTCF day 7 AHDC1/Gibbin KO rep 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
CTCF
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC H9
NA
NA
Attributes by original data submitter
Sample
source_name
H9 day7
genotype
AHDC1/Gibbin KO
chip antibody
anti-CTCF antibody produced in rabbit, Active Motif, CAT# 61311; RRID:AB_2614975
tag
N/A
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Briefly, cells were detached with Accutase and crosslinked in solution for 10 minutes in freshly prepared 1% formaldehyde (Thermo Scientific) in PBS. For Gibbin ChIP-seq, freshly collected cells were treated with 5% 1,6-hexanediol in 5mL PBS in suspension for 60 seconds, upon which the solution was immediately diluted with 25mL PBS and 2mL 16% formaldehyde for crosslinking. Cell were quenched in 0.125M glycine, followed by two washes with cold PBS. 25x106 cross-linked cells were used per ChIP replicate. Cells were lysed in lysis buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 0.5% SDS, 1X protease inhibitors) for 30 minutes on ice and sonicated twice for 10 minutes on a Diagenode Bioruptor to achieve a chromatin between 200 and 400bp in size. Chromatin was centrifuged, quantified and diluted in dilution buffer up to 2mL (50mM Tris-HCl pH 8.0, 10mM EDTA, 1X protease inhibitors). Sheared chromatin was incubated overnight at 4°C with a each antibody. The next day lysates were incubated with 30μL of Dynabead protein G beads (Invitrogen) for 4 hours at 4°C. Beads were washed twice each with low salt buffer (50mM Tris-HCl pH 8.0, 0.15M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), high salt buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), and LiCl buffer (50mM Tris-HCl pH 8.0, 0.15M LiCl, 1mM EDTA pH 8.0, 1% Nonidet P-40, 0.1% sodium deoxycholate). DNA was eluted in 100μL of elution buffer (50mM NaHCO3, 1% SDS) and crosslinks were reversed with 4μL of 5M NaCl incubated shaking for 16 hours at 67°C. RNA was removed by adding 1μL of 10mg/mL RNase A for 30 minutes at 37°C. DNA was cleaned using the Qiaquick PCR purification kit (Qiagen) and quantified using Qubit (Invitrogen). 10ng of DNA was used to make libraries with the NEBNext kit (New England Biolabs) and AMPure XP beads (Beckman Coulter) according to the manufacturer instructions.