GSM2262750: ere_3L_1; Drosophila erecta; RNA-Seq
Dissected tissue was stored in Trizol reagent for pooling. RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s instructions, including an on-column DNase digestion (Qiagen). RNA concentrations were measured and 700ng RNA was diluted to 60ul total volume with H2O. RNA sequencing libraries were prepared with TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to manufacturer’s instructions. For the RNA fragmentation step, 94˚C, 2min was used with the intention to obtain a median size ~185bp. PCR amplification was done with 15 cycles. A total of 24 multiplexed libraries (barcoded) were accessed for quality and combined together before separation into two identical pooled libraries, which were then subjected to cluster generation followed by Illumina 50bp paired-end sequencing by the UNC High-Throughput Sequencing Facility (HTSF).