title: Illumina HiSeq 2000 paired end sequencing; Transcriptome of Venturia canescens VLP-producing tissue (calyx)
design: A cDNA library was generated from total RNA extracted from the calyx of thirty 24-hour-old females from the wasp thelytokous laboratory strain. The mRNA was converted to cDNA with the Illumina TruSeq RNA sample preparation kit. Paired-end sequencing (2x100-bp reads) was performed on an Illumina HiSEQ2000 sequencer at the Genoscope national sequencing center. Illumina PE reads were cleaned by a three-step procedure: i) sequencing adapters and low-quality nucleotides (quality value lower 20) were removed, ii) sequences between the second unknown nucleotide (N) and the end of the read were removed, iii) reads shorter than 30 nucleotides after trimming were discarded, together with reads and their mates that mapped onto run quality control sequences (PhiX genome).
library_construction_protocol: Calyx of 30 fresh thelytokous females aged of 24h were dissected in a phosphate-buffered-saline 1X solution and immediately transferred in 200uL of RLT buffer from the RNeasy Mini kit (Qiagen). Total RNA was extracted according to manufacturer’s protocol and contaminating DNA was removed with Turbo-DNA free kit (Ambion), following manufacturer’s instructions. A cDNA library was generated from 2.17ug total RNA using TruSeq RNA Sample Preparation Kit-Set A (Illumina), according to manufacturer’s instructions.