GSM6621040: induced MCF10A YAP5SA cells p63; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
TP63
Cell type
Cell type Class
Breast
Cell type
MCF 10A
Primary Tissue
Breast
Tissue Diagnosis
Fibrocystic Disease
Attributes by original data submitter
Sample
source_name
MCF10A-YAP5SA
chip antibody
p63-alpha (D2K8X) (Cell Signalling, #13109)
cell line
MCF10A-YAP5SA
Sequenced DNA Library
library_name
GSM6621040
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-seq, cells were cross-linked with 1% formaldehyde (Sigma) for 10 min at room temperature. The reaction was stopped by adding 125 mM glycine (Sigma). After cells were lysed for 10 minutes on ice [5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40, 1 mM PMSF, protease inhibitor cocktail (Sigma)], nuclei were resuspended in RIPA buffer [50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitor cocktail (Sigma)]. Chromatin was fragmented to an approximate length of 150 to 300 bp using a Branson sonifier. Antibodies (9 µg) were coupled to protein G dynabeads (Thermo Fisher Scientific) for 6 hours at 4°C and then incubated with fragmented chromatin over night at 4°C. Beads were washed in total twelve times with wash buffer I (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), wash buffer II (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), wash buffer III (50mM Tris-HCl pH8, 0.5M LiCl2, 1mM EDTA, 1% Nonidet P-40, 0.7% sodium deoxycholate) and wash buffer IV (10mM Tris-HCl pH8, 1mM EDTA). 1mM PMSF and protease inhibitor cocktail (Sigma ) was added freshly to all buffers. Chromatin was eluted in (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA, 0.5% SDS, 10µg/ml RNaseA) and crosslink was reversed at 65°C over night. Proteins were digested by adding 200 µg/ml proteinase K at 55°C for 2 hours. DNA was purified using the QIAquick PCR Purification Kit (QIAGEN) and eluted in 50 µl EB buffer. For ChIPseq, DNA libraries were generated using 10ng purified ChIP-DNA and the NEBNext®Ultra II DNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer's instructions. DNA library was amplified by 15-18 PCR cycles and quality was analyzed using the Fragment Analyzer (Advanced Analytical). Libraries were sequenced on the NextSeq 500 platform (Illumina).