GSM7008817: SUM149 WT RelB ChIP Rep3; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RELB
Cell type
Cell type Class
Breast
Cell type
SUM 149PT
Primary Tissue
Breast
Tissue Diagnosis
Adenocarcinoma Ductal
Attributes by original data submitter
Sample
source_name
SUM149
cell line
SUM149
treatment
WT
replicate
Rep3
chip antibody
RelB
geo_loc_name
missing
collection_date
missing
Sequenced DNA Library
library_name
GSM7008817
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP was performed with an adapted disuccinimidyl glutarate (DSG) and formaldehyde chromatin crosslinking protocol (Wang 2022, Tian 2012, Singh 2019). 2-5x106 cells were collected in PBS, fixed with 2mM DSG reagent (Thermo Fisher Scientific) for 45min rotating at room temperature, washed, fixed with 1% formaldehyde (Thermo Fisher Scientific) for 10min rotating at room temperature, and quenched with 125mM glycine rotating for 5min at room temperature. Fixed cells were lysed in Buffer 1 (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, freshly supplemented protease and phosphatase inhibitors) by rotation for 10min at 4ºC, pelleted for 5min at 3000rpm at 4ºC, lysed in Buffer 2 (10mM Tris-HCl pH8, 200mM NaCl, 1mM EDTA pH8, 0.5mM EGTA, freshly supplemented protease and phosphatase inhibitors) by rotation for 10min at 4ºC, pelleted for 5min at 3000rpm at 4ºC, and resuspended in Buffer 3 (10mM Tris-HCl pH8, 100mM NaCl, 1mM EDTA pH8, 0.5mM EGTA, 0.1% NaDOC, 0.5% N-laurylsarcosine, freshly supplemented protease and phosphatase inhibitors) for sonication. Cells were sonicated on High for 30min with 30sec on/off in a water bath sonicator maintained at 4ºC (Diagenode). 10% Triton X-100 was added to sonicated lysates and spun at 11,000rpm for 10min at 4ºC to pellet debris; 5% input and the appropriate number of aliquots were taken from sonicated lysate. Following overnight antibody incubation and incubation with BSA-blocked Dynabeads (beads washed in RIPA buffer were incubated with 0.1ug/uL BSA for 30min followed by RIPA wash) for 1h at room temperature, beads were sequentially washed in low salt (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS), high salt (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS), and LiCl (250mM LiCl, 10mM Tris-HCl pH8, 1mM EDTA pH8, 1% NP-40, 1% NaDOC) buffers. DNA was eluted with Elution buffer (1% SDS, 100mM NaHCO3) and agitation for 15min at 42ºC. ChIP and input DNA was reverse crosslinked with RNaseA (0.2mg/mL) and NaCl (20mM) for 2h at 65ºC and Proteinase K (0.4mg/mL) for 1h at 50ºC before processing using Qiagen PCR Clean Up Kit per kit protocol ChIP-seq libraries (Illumina) were prepared according to manufacturer's recommendations and sequenced at the UNC High Throughput Genomic Sequencing facility using paired-end 50bp reads