title: Illumina NovaSeq 6000 paired end sequencing; Genome-wide binding signature of Signal transducer and activator of transcription (STAT) family in colorectal cancer HCT 116 cell
design: Genome-wide binding signature of Signal transducer and activator of transcription (STAT) family in colorectal cancer HCT 116 cell
library_construction_protocol: 1x10^7 HCT116 cells are cultured within DMEM containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). The siRNA (UCAUUGAGCAAAAGAAAAGUG) is used to silence NR5A2 by lipofectamin 3000 (Thermo Fisher Scientific). 48 h later, cells transfected by siRNA can be harvested for the consequent experiments. 1x10^7 HCT116 cells are directly scraped from dish, and centrifuged by 800 rpm for 5 min in 4 oC. The supernatant is removed. Cells are washed by PBS twice, and resuspended by 1 mL RIPA buffer, and put on ice for 30 min, followed by highest speed centrifugation for 15 min. The supernant containing with nucleus are transfered to another new tube. Whole cell lysates are sonicated to break up the genomic DNA into 200-500 bp fragments. Then, 10% lysates were saved as input and the remaining were incubated with 1 g IP grade antibodies of STAT1 (sc-464, Santa Cruz Biotechnology, Santa Cruz, CA, USA), STAT2 (sc-514193, Santa Cruz Biotechnology), STAT3 (sc-293151, Santa Cruz Biotechnology), STAT5A/B (sc-74442, Santa Cruz Biotechnology) or STAT6 (sc-374021, Santa Cruz Biotechnology) at 4oC overnight. This is followed by incubating for 2 h with Protein A beads (Thermo Fisher Scientific) at 37 C to pull down the bound DNA fragments. DNA fragments from the pull-down is isolated by ethanol precipitation.For high throughput sequencing, we added 3-dA overhang to the STATs enriched or input DNA and ligated them to the adapter to build a DNA library.