GSM6080981: HCT116 cells, Wild-type transposon with re-engineered piggyBac replicate 3; Homo sapiens; OTHER
RNA was extracted using RNaesy Mini Kit (Qiagen #74106). Total RNA was reverse-transcribed to cDNA using M-MLV (Promega #M3681) and primer SMART-dT18VN (5`-AAGCAGTGGTATCAACGCAGAGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN). Next, cDNA was amplified using different pairs of primers containing regions within specific tranpsoson used in this study (see suplemental information) and primer Smart (5`-AAGCAGTGGTATCAACGCAGAGT). Amplified cDNA was purified using PCR gel purification column (Macherey-Nagel #740609) and 500 ng of purified cDNA was was fragmented and tagged using Illumina DNA Prep (Illumina #20060060). Tagged DNA was PCR amplified (Read1-TnME: 5`-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGATGTGTATAAGAGACA*G; Read2 primer was specific to transposase species, see suplemental information) and amplicons from 100-500 bp were size selected on 2% agarose gel. Dual index primers (NEB #E7780S) for seqeuncing were added by amplifying tagmented libraries usign Q5 Polymerase (NEB #M0544S) following the NEBnext protocol (NEB #E7645S - Section 4.1). The PCR products were purified using AMPure XP beads (Beckman Coulter #A63881) using 0.9x bead volume. The quality of final library was verified using TapeStation. See the publication 10.1016/j.cell.2020.06.037 for detailed methods to construct library from self-reporting transposons (SRTs).