NextSeq 500 paired end sequencing; Cell-type-specific modulation of longevity by Forkhead family transcription factors in the Drosophila nervous system
Total RNA from fly heads (30 heads per sample) was extracted using the QIAGEN total RNA isolation kit and quantified on an Agilent 2100 Bioanalyzer. Sample concentration and purity of RNA was measured on a NanoDrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer.  RNA was extracted using the QIAGEN total RNA isolation kit and quantified on an Agilent 2100 bioanalyser. Sample concentration and purity of RNA was measured on a NanoDrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer. Samples were processed using Illumina's TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to manufacturer's instructions.  Deviations from the protocol were as follows:250ng total RNA was used as starting material.  Fragmentation was carried out for 10mins instead of 8mins, and 14 cycles of PCR were used.mRNA was isolated from total RNA using Oligo dT beads to pull down Poly-Adenylated transcripts. The purified mRNA was fragmented using chemical fragmentation (heat and divalent metal cation) and primed with random hexamers.  Strand-specific first strand cDNA was generated using Reverse Transcriptase and Actinomycin D. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. Full length TruSeq adaptors, containing a T overhang were ligated to the A-Tailed cDNA. Successfully ligated fragments were enriched with 14 cycles of PCR.