title: RNA seq of Athalia rosae: whole male L4 larvae
design: Novogene (Cambridge, UK) carried out library preparation (with a Ribo-Zero for rRNA removal) see below for details from report:After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. The mRNA is then fragmented randomly by adding fragmentation buffer. The cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. The quality control of library consists of three steps: 1) Qubit 2.0: tests the library concentration preliminarily, 2) Agilent 2100: tests the insert size, 3) Q-PCR: quantifies the library effective concentration precisely. The qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume.