Enrichment and sequencing of SARS-CoV-2 infected Calu-3 cell DNA
Tn5 tagmentation mediated integration site enrichment sequencing. Cellullar DNA extraction was done using a published method (Lee et al. Nature, 563, 639–645(2018)). We used a tagmentation based method to enrich for viral integration sites (doi: https://doi.org/10.1101/037762). Briefly, we used Tn5 transposase (Diagenode C01070010) to randomly tagment the cellular DNA with adapters (adapter A, the Illumina Nextera system). Tagmentation was done using 100 ng DNA for 10 min at 55 °C, followed by stripping off the Tn5 transposase from the DNA with SDS. We used a reverse primer (CCAAGACGCAGTATTATTGGGTAAA) targeting the viral NC gene to linearly amplify (PCR0, 45 cycles) the tagmented DNA fragments containing viral sequences. We took the product of PCR0 and amplified the DNA fragments containing adapter and viral NC sequences (potential integration sites) using 15-20 cycles of PCR1, with a barcoded (i5) Nextera primer (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC) against the adapter sequence and a primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCGACGTTGTTTTGATCG) that was designed to be close to the near 5' end of viral NC gene (GCCGACGTTGTTTTGATCG) and contained another adapter sequence. We further amplified the PCR1 product by 15-20 cycles of PCR2, using a short primer (AATGATACGGCGACCACCGA) against the i5 Nextera primer sequence and a barcoded (i7) Nextera primer (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG) against the adapter sequence introduced by the viral primer in PCR1. The final product of the PCR2 amplification was fractionated on 1.5% agarose gel (Sage Science HTC1510) with PippinHT (Sage Science HTP0001) and 500 – 1000 bp pieces were selected for Illumina paired-end sequencing. All three PCR steps (PCR0 – PCR2) were done with KAPA HiFi HotStart ReadyMix (KAPA KK2602).
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