GSM8499202: Pupa day 6, rep 3; Bicyclus anynana; OTHER
We extracted both DNA and RNA from the same individual butterfly sample. Disruption and homogenization of tissue were performed using TissueLyser (Qiagen) as per the manufacturer's instructions. Briefly, 1mL of cold PBS was added directly to each of the samples with autoclaved 3mm stainless steel beads and disruptions were carried out gently at 20Hz for 1 minute. To perform simultaneous RNA and DNA extractions using QIAzol and ATL buffer, we immediately divided the homogenate after homogenization, allocating portions for DNA extraction with the Qiagen DNeasy kit (Cat. No. 69504) and RNA extraction using the RNeasy Mini Kit (Cat. No. 74104). As described below, each suspension was subjected to a further cycle of homogenization at 20Hz for 30 seconds in the respective DNA or RNA extraction lysis buffers. DNA was extracted as per the manufacturer's instructions using the Qiagen DNeasy® Blood & Tissue Kit with the following modifications: after addition of the lysis buffer an additional homogenization cycle was performed for 30 seconds at 20Hz; centrifugation was performed at 17,000 x g for three minutes instead of 20,000 x g; and 100 µl of buffer AE was used during the elution step instead of 200µL. The final eluate was stored at −80°C. DNA libraries were prepared from 1 mg aliquots of genomic DNA using the Oxford Nanopore Technology Native Barcoding Kit 24 V14 (SQK-NBD114.24) following the manufacturer's protocol.