GSM4103427: Pooled control RNA-seq; Danio rerio; RNA-Seq
Total RNA was extracted from three different batches of 100 livers of uninjected control and man1a2 MO-injected larvae at 5 days post fertilization by using RNeasy Mini Kit. The pooled RNA was used to generate mRNA sequencing libraries using Illumina TruSeq Stranded mRNA sample preparation kit. Poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads, mRNA fragmented into small pieces using divalent cations, and copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were ligated to single 'A' base and adapter, then purified and enriched with PCR to create the final cDNA libraries.