title: GSM6282843: WT, 25, Rep3; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For the wing, disc-associated myoblasts dissociation was performed according to a modified protocol (Khan et al., 2016). 0 h APF Mef2-GAL4, UAS-GFP::Gma/WT or UAS-M1BP RNAi #1 expressing larvae were washed in PBS, sterilised with 70% ethanol and dissected in Supplemented Schneider's Medium (SSM) containing 10% Foetal bovine serum, 2% Penicillin/Streptomycin and 0.02 mg/mL Insulin. 30 to 35 wing discs were collected in ice-cold PBS and dissociated using preheated TrypLE 10x at 30°C for 45 min at 300rpm. The reaction was stopped by adding SSM and the cells were mechanically resuspended by gentle pipetting. Aggregates were removed by filtering cells in a cell strainer (30µm, Miltenyi) and cells were sorted using a FACS AriaII Machine (BD), according to cell viability, size and GFP intensity. Myoblasts were sorted directly in the kit-provided Lysis buffer supplemented with 2-Mercaptoethanol (#M6250, Sigma) and kept at -80°C until RNA extraction, which was preferentially performed the same day. RNA was isolated from the pure myoblast population of approximately 50 000 cells using RNA extraction Microkit (#74004, Qiagen). Full length cDNA were generated from 2ng of total RNA using SMART-SeqX v4 UltraX Low Input RNA Kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 10 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundreds pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, USA) followed by 12 cycles of library amplification. Following purification with SPRIselect beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoreris.