title: GSM9462945: Cavia porcellus cochlear, with cisplatin treatment, rep3; Cavia porcellus; RNA-Seq
library_construction_protocol: RNA was harvested using Rneasy mini plus kit (Qiagen).1ug of total RNA was used for the construction of sequencing libraries For mRNA enrichmcnt, poly(A) mRNA isolation was performed using Oligo(dT) beads. Alternatively, when the sample quality or experimental design necessitated, ribosomal RNA was depleted using rRNA removal methods to enrich for coding and non-polyadenylated transcripts. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA synthcsis was carricd out using reverse transcriptase, followed by second-strand cDNA synthesis. For strand-specific library construction, during the second-strand synthesis the standard dTTP was replaced with dUTP; subsequent treatment with USER enzymc selectively degrades the second strand prior to PCR amplification, thereby preserving strand information. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tail in a single reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers, and thc PCR products werc validated. Finally, libraries with different indexes were multiplexed and loaded on an Illumina HiSeg/ Illumina Novaseq/ MG12000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to the manufacturer's instructions.