GSM4887218: NIM TCDD ChIP-seq AHR rep1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
AHR
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC derived neural cells
NA
NA
Attributes by original data submitter
Sample
source_name
neural progenitors
cell type
hESC H9 derived neural progenitors
cell line
H9
chip antibody
anti-AHR antibody (SA-210)
treatment
TCDD
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Prior ChIP cells were treated with DMSO or 100 nM TCDD at 37oC for 1.5 h.1% formaldehyde was used for cross-linking the cells at RT for 10 min. Cross-linking was stopped with glycin at RT for 5 min. Following steps until eluation were performed at 4oC. Cells were washed twice with cold PBS, ~10M cells per sample were harvested by scraping and centrifuged at 1000g for 5 min. For nuclei isolation, pellet was resuspended in 250 µl Nuclei Lysis Buffer 1 (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25% Triton X-100, 10 mM KCl) and incubated for 10 min with gentle agitation. Following centrifugation at 1350g for 5 min, pellet was resuspended in 250 µl Nuclei Lysis Buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated for 10 min with gentle agitation. Nuclei were centrifuged at 1350g for 5 min and pellet resuspended in 100 µl SDS Lysis buffer (1% SDS, 20 mM EDTA, 50 mM Tris-HCl pH 8.0, Protease inhibitor cocktail). Lysis was performed on ice for 10 min and chromatin was sheared to appropriate length fragments using BioRuptor Plus sonicator (Diagenode) (10 cycles of 30 sec ON/OFF). Lysates were then diluted with 10X ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, Protease inhibitor cocktail) to total volume 1ml and centrifuged at 10000g for 10 min. Supernatant was collected, 1% was frozen for Input control and 500 µl sample was incubated overnight with 8 ug anti-AHR antibody (SA-210). Lysates were incubated for 2 h with Protein G Dynabeads (Thermo Scientific). Beads were washed for 5 min with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), LiCl buffer (0,25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Tube switch was performed in between last two washes to enhance specificity [71]. Chromatin-antibody complexes were eluted from Dynabeads twice with 250 µl of elution buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS) at 65oC for 15 minutes on a rotator and eluates were combined. NaCl at final concentration of 300 mM was added and tubes were incubated overnight at 65oC. Proteins were digested using 40 µg/ml Proteinase K (Thermo Scientific) at 50oC for 2 h and RNA digested using 20 µg/ml RNase A (Thermo Scientific) at 37oC for 30 min. DNA was purified using FavorPrep™ PCR Clean-UP Kit (Favorgen). Immunoprecipitated and input DNA was used for library preparation with Ovation Ultralow Library System Kit V2 (NuGen) according to manufacturer's instructions.