GSM7844589: HTC116 cells, WT, 2; Homo sapiens; OTHER
Total RNA was extracted from HCT116 cells using NucleoSpin RNA Plus (Macherey-Nagel) and reversed transcribed targetting polyA RNA with primer SMART-dT18VN, 5'-AAGCAGTGGTATCAACGCAGAGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' cDNAs were PCR amplified with four primers located in transposon areas, SRT-PAC-F1, 5'-CAACCTCCCCTTCTACGAGC-3', SRT-Seq P1, 5'-CGGTGCCTGATAACAACTCG-3', SRT-Seq P2, 5'-GGTTCAGCAGGAATGCCGAG-3', and SRT-Seq P3 5'-CTCAGTGTCCATAGCGACGC-3' and one primer located in Smart-dT18VN, Smart: 5'-AAGCAGTGGTATCAACGCAGAGT-3' Amplicons were fragmented with and tagged with Illumina DNA Prep (Illumina). Tagged DNA fragments were further PCR amplified using Read1-TnME, 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGATGTGTATAAGAGACA*G-3', and Read2-Pbat, 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTTTCCCGCAATCCAAGTG -3', containing sequencing adapters, to enrich for fragments carrying the LE end of the transposon and downstream genomic insertion site PCR amplicons were 100-500bp size selected with 2% agarose gel before sequencing library preparation NEBNext Ultra II Q5 Master Mix (NEB, M0544S) was used to barcode size-selected amplicons for Illumina sequencing (NEB, E7780S). The amplified fragments were purified and size selected using AMPure XP beads (Beckman Coulter, A63881) at 0.9x bead/sample ratio.