GSM7689473
Genomic locations of ORFtag integrations were mapped using modified inverse-PCR followed by next generation sequencing (iPCR-NGS) protocol8. Genomic DNA was prepared by lysing cell pellets in lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K) at 55°C overnight. Following a 2-hour RNase A treatment (Qiagen, 100 mg/ml, 1:1,000 dilution) at 37°C, two extractions using phenol:chloroform:isoamyl alcohol and one extraction using chloroform:isoamyl alcohol were carried out. The samples then underwent two separate digestion reactions (with up to 4 µg of genomic DNA) using NlaIII and MseI enzymes (NEB) at 37°C overnight, followed by purification using a Monarch PCR&DNA Cleanup Kit (NEB). Ring-ligation was carried out using T4 DNA ligase (NEB) at 16 °C overnight, followed by heat-inactivation (65°C, 15 min) and linearization using SbfI-HF (NEB) at 37°C for 2 h. The digests were then purified using a Monarch PCR&DNA Cleanup Kit (NEB) and amplified using firstly a nested PCR reaction with KAPA HiFi HotStart ReadyMix (Roche), and a specific primer pair (TGCAGGACCGGACGTGACTGGAGTTC*A, TGCAGGACGATGAGCAGAGCCAGAACC*A) for 16 cycles. After cleanup with AMPure XP Reagent (Beckman Coulter, 1:1 ratio beads:PCR), iPCR amplification was carried out with KAPA HiFi HotStart ReadyMix (Roche), and a specific primer pair (AATGATACGGCGACCACCGAGATCTACACGAGCCAGAACCAGAAGGAACTTGA*C, CAAGCAGAAGACGGCATACGAGAT [custom-barcode] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) for 18 cycles. Afterwards, amplified libraries were size selected for a range of 400-800 bp using SPRIselect beads (Beckman Coulter). NGS was performed on an Illumina NextSeq550 or llumina HiSeq 2500 sequencer according to the manufacturers' protocols with custom first-read primer (1:1 mix of GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA and TGAGTGATTGACTACCCACGACGGGGGTCTTTCA).