GSM2365668: Dpse_21; Drosophila pseudoobscura; RNA-Seq
Total RNA was extracted from embryos using a Beadbeater (Biospec, Cat. #607) with 1.0 mm zirconia beads (Biospec, #11079110zx) and the RNAdvance Tissue kit (Agencourt #A32649) according to the manufacturer’s instructions, including DNaseI treatment. We systematically checked on a Bioanalyzer RNA Nano chip (Agilent) that the RNA was of very high quality. Libraries were prepared as described in Batut et al., Genome Research 2012. In brief, 5’-monophosphate transcripts were depleted by TEX digest (Epicentre #TER51020). For every time series, each sample was labeled with a different sequence barcode during reverse-transcription, and all samples for the series were then pooled and processed together as a single library. The 5'-complete cDNA selection strategy relies on the combination of two orthogonal enrichment methods: reverse-transcriptase template-switching, and cap-trapping. The template-switching approach is based on the ability of reverse-transcriptase to add linker sequences to the ends of 5'-complete cDNAs – preferentially if they are made from capped transcripts. Cap-trapping relies on the biotinylation of capped RNA molecules and specific pulldown of their associated 5'-complete cDNAs. Quality control and library quantification were carried out on a Bioanalyzer DNA High Sensitivity chip. Each library was sequenced on one lane of an Illumina HiSeq 2000. reference: Batut, P., Dobin, A., Plessy, C., Carninci, P. & Gingeras, T.R. High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. Genome Res (2012). reference: Batut, P. & Gingeras, T.R. RAMPAGE: Promoter Activity Profiling by Paired-End Sequencing of 5'-Complete cDNAs. Curr Protoc Mol Biol 104, 25B 11 1-25B 11 16 (2013).