GSM2411203: Virgin Berlin rep 1; Drosophila melanogaster; RNA-Seq
Flies were flash frozen and heads were separated from the bodies. Total RNA was extracted from the heads using Trizol, mRNA was isolated using MicroPoly(A) Purist columns (Ambion). After adding the spike-in control RNAs, samples were fragmented to ∼250 bp by chemical fragmentation. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and a combination of random hexamer and oligo (dT) primers; second-strand cDNA was synthesized using DNA polymerase I in combination with ribonuclease H. Double-stranded cDNA templates were blunt ended using End-It Repair kit (Epicentre), and A-overhangs were added at both ends with Klenow fragment (3′→5′ exo-minus). Illumina GAII sequencing adaptors were then ligated to both ends of the cDNA templates using Fast-Link DNA ligation kit (Epicentre). We then enriched for cDNA templates by performing multiplex incorporating PCR reactions (18 cycles) and isolated 250- to 550-bp fragments by gel purification