GSM3941235: CFPAC1.FOXA2KO.ChIP.FOXA2.Rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
CFPAC1 genome-edited clonal cell line
Pancreatic Ductal Adenocarcinoma (PDAC) cell line
Anti-FOXA2 (Antibody sc-6554 Lot. A1014)
Isolation of clonal cell line by dilution of CFPAC1 cells electroporated with plasmid pX330A_D10A-1x4 containing Cas9 nickase (Multiplex CRISPR/Cas9 Assembly System Kit; Addgene # 1000000055) and guides targeting FOXA2 gene.
Sequenced DNA Library
ChIP lysates were generated from 50x10^6 cells. Cells were fixed in 1% formaldehyde for 10min. Lysate was immunoprecipitated with 10ug of antibody. Antibodies were pre-bound overnight to 100ul of G protein-coupled paramagnetic beads in PBS/BSA 0.5%. Beads were then added to lysates (the preclearing step was omitted) and incubation was let to proceed overnight. Beads were washed 6 times in a modified RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in TE containing 50mM NaCl. DNA was eluted in TE containing 2% SDS and crosslinks reversed by incubation overnight at 65ºC. DNA was then purified by AMPure XP SPRI (Beckman Coulter) and quantified using Quantifluor (Promega). ChIP DNA was prepared for HiSeq2000 or NextSeq500 Illumina sequencing using a standard protocol consisting in blunting, addition of dA overhangs, ligation of Illumina adapters, PCR with index primers and purification. A mixture of T4 DNA polymerase, DNA polymerase I and T4 kinase was used according to manufacturer's instruction. Library preparation is carried out on SPRIworks Fragment Library System.