GSM5620871: Control_sample_rep1; Drosophila melanogaster; RNA-Seq
Six days after egg-laying, as larvae began to crawl out of the food to proceed with pupae formation, individual third-instar larvae were collected using a needle, transferred to microcentrifuge tubes, and frozen at -80°C for 24 to 48 hours. Freezing was followed by the addition of 200uL of RNAlater-ICE Frozen Tissue Transition Solution (AM7030, Thermo Fisher Scientific) to larvae. Approximately 30 larval brains were dissected per replicate in RNAlater-ICE, for a total of three replicates (~90 larval brains) for each group. Total RNA was extracted from larval brains using EZ1 RNA Tissue mini kit (EZ1 RNA Tissue Mini Kit no. 959034, Qiagen). All samples were treated with DNase I (DNase Set no. 79254, Qiagen). RNA concentration and quality were evaluated using a Qubit 2.0 fluorometer (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Capture of mRNA (poly-A selection) and library preparation was carried out using the Illumina TruSeq® Stranded mRNA Library Prep kit from 300ng of total RNA. Libraries were sequenced with an Illumina NextSeq 500/550 High Output Kit v2.5 for 80 cycles following manufacturer's protocol (Illumina, Inc). At least 20 million reads were obtained for each replicate (a pool of 30 brains).