GSM3508096: 22rv1-crispr-#wt3-foxa1-tfs; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Sequenced DNA Library
8-10 million cells ectopically overexpressing different V5-tagged FOXA1 variants and WT AR (or TLE3) were fractionated to isolated intact nuclei using the NE-PER kit reagents (Thermo Fisher Scientific; Cat#: 78835) and lysed in the complete IP lysis buffer (Thermo Fisher Scientific; Cat#: 87788). Nuclear lysates were incubated for 2 hours at 4C with 30ul of magnetic Protein-G Dynabeads (Thermo Fisher Scientific; Cat#: 10004D) for pre-clearing. A fraction of the cleared lysate was saved as input and the remainder was incubated overnight (12-16 hours) with 10ug of target protein antibody at 4C with gentle mixing. Next day, 50ul of Dynabeads Protein-G beads were added to the lysate-antibody mixture and incubated for 2h at 4C. Beads were washed 3 times with IP buffer (150nM NaCl; Thermo Fisher Scientific) and directly boiled in 1X NuPage LDS/reducing agent buffer (ThermoFisher Scientific; Cat#: NP0007 and NP0009) to elute and denature the precipitated proteins. These samples were then immunoblotted as described above with the exception of using protein A-HRP secondary (GE HealthCare, Cat#: NA9120-1ML) antibody for detection. The ChIP-seq sample preparation for sequencing was performed according to the manufacturer's instructions (Illumina). ChIP-enriched DNA samples (1-10 ng) were converted to blunt-ended fragments using T4 DNA polymerase, E.coli DNA polymerase I large fragment (Klenow polymerase) and T4 polynuleotide kinase (New England BioLabs, NEB). A single A-base was added to fragment ends by Klenow fragment (3' to 5' exo minus; NEB) followed by ligation of Illumina adaptors (Quick ligase, NEB). The adaptor-modified DNA fragments were enriched by PCR using the Illumina Barcode primers and Phusion DNA polymerase (NEB). PCR products were size selected using 3% NuSieve agarose gels (Lonza) followed by gel extraction using QIAEX II reagents (QIAGEN). Libraries were quantified with the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina HiSeq 2000 Sequencer (100 nucleotide read length).