RNA -seq of Crocuta crocuta: adult female blood
A total of 1.1 g of each RNA sample was used as input as required by the workflow. The preparation of dual-indexed, sequencing libraries entailed the following: 1) The purification of poly(A) mRNA molecules using poly(T) oligo-attached magnetic beads. 2) The fragmentation of RNA, followed by priming with random hexamers for cDNA synthesis. 3) The reverse transcription of the cleaved and primers RNA fragments into first-strand cDNA using reverse transcriptase and random primers. 4) The removal of the RNA template and synthesis of a replacement strand, incorporating deoxyuridine triphosphate (dUTP) in place of deoxythymdine triphosphate (dTTP) to generate double-stranded cDNA (ds cDNA). 5) The addition of an A nucleotide to the 3-ends of the blunt fragments. 6) The ligation of indexing adapters with a single T nucleotide to the ends of the ds cDNA, preparing them for hybridization onto a flow cell. This included the attachment of unique dual indices to all for multiplexing of the samples. The selective enrichment and amplification of DNA fragments with adapter molecules on both ends using PCR.