GSM2344916: MiaPaCa2.delta-Repeat.ZEB1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Pancreatic Ductal Adenocarcinoma (PDAC) cell line
MiaPaCa2 genome-edited clonal cell line
Anti-ZEB1 (Antibody SC-25388 Lot. K1615)
Isolation of clonal cell line by dilution of MiaPaca2 cells electroporated with two plasmids pX330A_D10A-1x2 driving the expression of the Cas9-nickase and different pairs of gRNAs designed to target the repeated genomic region upstream MIR200A/B locus . (Multiplex CRISPR/Cas9 Assembly System Kit).
Sequenced DNA Library
ChIP lysates were generated from 20-40x10^6 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 QIAquick columns (Qiagen) and quantified using PicoGreen (Invitrogen). ChIP DNA was prepared for HiSeq 2000 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.