title: Whole genome sequencing of EMS-treated Wolbachia-infected (wMel) JW18 Drosophila cells
design: Genomic DNA was purified from cell pellets by centrifugation at 10,000 x g for 3 min at 4C. DNA was sheared by sonication (30% duty cycle, 60W peak power, 1000 cycles/burst, two 5-min rounds), phosphorylated with T4 PNK, denatured, and circularized with no more than 3.5 pmol ssDNA. Linear DNA was digested and rolling circle amplification (RCA) was performed using 2.5 ul of 10 uM random hexamer primers and 2.5 ul of 10 mM dNTP. RCA products were treated with Exonuclease I and III to remove linear fragments, then purified with SPRI magnetic beads. Libraries were prepared by tagmentation of 10 ng RCA product with in-house Tn5 transposase in TAPS-PEG 8000 buffer (8 min at 55C), followed by 12-cycle PCR amplification with KAPA HiFi Polymerase and unique dual index primers. PCR products were size-selected at 400-700 bp by gel extraction. Libraries were pooled and sequenced using 150-bp paired-end reads across two Illumina HiSeq 4000 lanes at Fulgent Genetics.