title: GSM6282825: WT, 48h APF, 2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Mef2-GAL4/WT or UAS-M1BP RNAi expressing pupa were staged at 25°C and dissected in ice-cold PBS. The dissection was performed according to a published protocol (Weitkunat and Schnorrer, 2014). Hand-isolated IFM from 6 to 8 pupa were directly collected in the kit-provided Lysis buffer supplemented with 2-Mercaptoethanol (#M6250, Sigma), homogenised with a small pestle and RNA was extracted using RNA extraction Microkit (#74004, Qiagen). P2637 : RNA-Seq libraries were generated according to manufacturer's instructions from 100 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94oC for 8 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 13 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.