GSM1974442: IS1; Oryctolagus cuniculus; RNA-Seq
Total RNA from uterine samples was extracted using TRIzol reagent (invitrogen). RNA purity was assessed using the ND-1000 Nanodrop and RNA integrity was evaluated using the Agilent 2200 TapeStation. The following RNA quality control parameters were used: A260/A280 ratio ≥ 1.8, A260/A230 ratio ≥ 2.0 and RIN value ≥ 7.0. The mRNA-Seq sample preparation kit (Illumina) was used for the preparation of RNA-Seq libraries. Briefly, poly-(A)+ mRNA was purified with oligo-(dT) magnetic beads and fragmented to approximately 200 bp in fragmentation buffer. The obtained cleaved RNA fragments were reverse transcribed to first-strand cDNA, followed by second-strand cDNA synthesis. The double-stranded cDNA fragments were purified, end-repaired and then ligated to sequencing adapters. After purification, suitable fragments were amplified through 15 cycles of PCR to generate the final sequencing libraries. Finally, PCR products were purified and quantified for high-throughput sequencing using the Illumina HiSeq™ 2500.