GSM2226527: REH Input ChIPseq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RAG1
Cell type
Cell type Class
Blood
Cell type
REH
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Lymphocytic
Attributes by original data submitter
Sample
source_name
REH
cell line
REH
chip antibody
RAG1
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For mouse thymocytes RAG1 ChIP-seq, cells were crosslinked with 1% HCHO, quenched with 0.125 M glycine, washed and resuspended in high-salt RIPA buffer (10 mM Tris pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.8 M NaCl, 1 mM PMSF, 1 µg/µL pepstatin A) and sonicated using a water bath sonicator (Diagenode) to obtain DNA of ˜ 100-250 bp.H3K27Ac ChIP-seq in REH cells was performed similarly, with the following modifications. After crosslinking, cells were resuspended in SDS Lysis Buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF, 1 µg/µL pepstatin A; 200 µls SDS Lysis Buffer per 10 million cells). For each immunoprecipitation, 300 µls of supernatant (corresponding to 15 million cells) was diluted to 1 mL in ChIP dilution buffer (167 mM NaCl, 16.7 mM Tris pH 8, 1.2mM EDTA, 1.1 % Triton X-100, 0.01% SDS) and pre-cleared with Protein G Dynabeads (Thermo-Fisher). The Dynabeads were then removed using a Dynal magnetic stand. After preclearing and adding blocking agent, anti-H3K27Ac antibody (5 µg; Abcam) was added, and the samples were incubated overnight at 4°C with rotation. Protein G Dynabeads were blocked with 2% BSA and 50 ng/µL heparin, and were then added to the samples, incubating for 2 hours at 4°C with rotation. The Dynabeads were washed (10 min, 4°C, with rotation, for each wash): 2x in Low Salt Wash Buffer (20mM Tris, pH 8, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS and 1 mM DTT), 2x in High Salt Wash Buffer (20mM Tris, pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 1 mM DTT), 2x LiCl Wash Buffer (10mM Tris pH 8, 1mM EDTA, 0.25 M LiCl, 0.5% NP-40, 1% sodium deoxycholate), 1x in TE + 0.2% Triton X-100, and 1x in TE. Beads were incubated twice with 150 µls of elution buffer (1% SDS, 0.1 M NaHCO3) for 15 minutes at room temperature. Eluates were pooled and incubated at 65°C overnight followed by treatment with RNase and Proteinase K. DNA was purified by standard phenol:chloroform extraction and ethanol precipitation. libraries were prepared and sequenced following standard Illumina protocols