Truseq single end rna sequencing protocols

Total RNA was extracted using TRIzol LS (Invitrogen) and the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) following the manufacturers' instructions as previously described 7 (link) . Silkworm fat bodies (30 mg) soaked in 300 μL TRIzol LS were homogenized. We isolated total RNA from the homoginates as previously described 7 (link) . The integrity of rRNA in each sample was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
5. 1. 5. Library preparation and sequencing: RNA-seq Sequencing was performed according to the TruSeq single-end RNA-sequencing protocols from Illumina for Solexa sequencing on a Genome Analyzer IIx with paired-end module (Illumina, San Diego, CA, USA). A total of 1 μg total RNA was used as the starting material for library construction, using the TruSeq RNA Sample Preparation Kit v2. This involved poly-A mRNA isolation, fragmentation, and cDNA synthesis before adapters were ligated to the products and ampli ed to produce a nal cDNA library. Approximately 400 million clusters were generated by the TruSeq SR Cluster Kit v2 on the Illumina cBot Cluster Generation System, and 36-65 base pairs were sequenced using reagents from the TruSeq SBS Kit v5 (all kits from Illumina). Short-read data were deposited in the DNA Data Bank of Japan (DDBJ)'s Short Read Archive under project ID DRA006123.