title: CAGE-seq of human colon cancer cell line (HCT116) upon TBX3 overexpression
design: CAGE-seq of human colon cancer cell line (HCT116) upon TBX3 overexpression
library_construction_protocol: Cells were cultured according to the growth protocol and harvested for RNA isolation and sequencing library construction. HCT116 human colorectal cancer cells were cultured in a 37 °C incubator in 5% CO2 and 89% humidity. Culture medium used was high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) supplemented with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco). Total RNA was isolated by using a standard TRIzol-based method (Cat#15596018, Thermo Scientific). The quality of the extracted RNA was tested on the Agilent 2100 Bioanalyzer. Each sample consisted of 5 μg of initial total RNA. Library preparation, including cap-trapping step and polyadenylation, was conducted according to a standard CAGE protocol (Noguchi et al., 2017). Briefly, RNA was reverse transcribed using random primers, followed by biotinylation of the RNA cap and 3' ends. Single-stranded, non-hybridized RNAs were digested with RNase leaving a 5' cDNA that was captured by streptavidin beads.