GSM1570731: nab-GAL4, UAS-prosperoRNAi, UAS-dicer2, UAS-chinmoRNAi BPC; Drosophila melanogaster; RNA-Seq
Wandering L3 VNCs were dissected in cold PBS 3 days after the 29°C switch, during 30-minute dissection rounds. Dissected VNCs were put in 500μL cold Lysis Buffer (RA1 from the Total RNA Isolation kit, Macherey Nagel) supplemented with 50 μL glass beads (diameter 0.75-1mm, Roth, A554.1) and frozen in liquid nitrogen at the end of the dissection round. Sample tubes were then stored at -80°C up to RNA extraction. Triplicates were made for each condition with brain numbers as follows (1) n=79, n=57, n=65; (2) n= 70, n=58, n=78; (3) n=51, n=60, n=82. For the RNA extraction, dissected brains stored in liquid nitrogen were thawed on ice. 10 μL TCEP were added to each tube following by 40s vortex. RNA extraction was then performed following the Total RNA Isolation NucleoSpin RNA XS protocol (Macherey Nagel). RNA quality and quantity was checked by running samples on an Experion RNA HighSens Chip (Biorad, 700-7105) and send to the Montpellier Genomix platforme for RNA sequencing. RNA-Seq libraries were prepared with the 'TruSeq Stranded RNA sample preparation Kit' (Illumina) according to the manufacturer's instructions. Stranded RNA-seq