GSM8499212: Larva 5th Instar, rep 1; Bicyclus anynana; RNA-Seq
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. The tissue homogenate was divided, not the tissue itself, to avoid any inconsistencies in mRNA profiling due to the uneven distribution of tissues and cell types. By splitting the tissue homogenate, we aim for a more random distribution of tissue types, ensuring consistent sampling. To prevent RNA degradation, we timed RNA extractions immediately after homogenization. RNA was extracted and purified using the QIAzol Lysis Reagent and Qiagen RNeasy Kits following the manufacturer's protocols. Briefly, 1mL of QIAzol was added to an aliquot of homogenate and an additional homogenization was performed for 30 seconds at 20Hz. The lysate was treated by addition of 0.3mL of chloroform per 1.3 mL of total lysate and the samples were then centrifuged at 12,000 x g for 15 minutes at 4°C. The supernatant was then removed to a clean tube and processed using Qiagen RNeasy spin columns according to the manufacturer's RNA purification protocol. DNase treatment was performed using RNase-Free DNase Set (74104, Qiagen). RNA was eluted using 25µL of RNase-free water and the eluate reapplied to the column for a second round of elution to generate a concentrated sample. The final eluate was stored at −80°C. RNA sequencing libraries representing full-length transcripts were prepared using the Oxford Nanopore Technology (ONT) PCR-cDNA Barcoding Kit (SQK-PCB111-24) according to the manufacturer's protocol.