GSM3597671: BJfibroblastsp63KLF4 InputChIP rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Epidermis
Cell type
BJ
Primary Tissue
Skin
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
BJ ATCC ® CRL-2522™
tissue
skin; foreskin normal
growth
cell culture, 37˚C
chip antibody
none
vector
p63KLF4
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Crosslinked cells were quenched with glycine (125mM final) for 5 min, followed by 2 x washes in cold PBS. Nuclei were then isolated from 20 million cells as previously described (Shah et at. 2013), and chromatin was sheared to 250bp average size using a Covaris S220. Immunoprecipitations were performed using 500µg sheared chromatin lysate and 5 µg antibodies preconjugated to Protein G beads (Invitrogen): H3K27ac (Active Motif, 39133), Flag (Sigma, M2, F1804) and hKLF4 (R&D Systems, AF3640). ChIP reactions were incubated for 16 hours at 4˚C with rotation and then washed 4x in wash buffer (50mM HEPES-HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-laurylsarcosine), followed by 1 wash in ChIP final wash buffer (1xTE, 50mM NaCl). Immunoprecipitated DNA was eluted from washed beads, reverse crosslinked overnight, purified and used to construct libraries. Sequencing libraries for ChIP experiments were prepared using NEBNext Ultra reagents (New England Biolabs). All ChIP samples and input were double-end sequenced on an Illumina NextSeq 500.