GSM6858766: Testis, in vitro D16, FT, rep1; Mus musculus; RNA-Seq
Total RNA was extracted from testicular samples using RNeasy Micro kit (74004; Qiagen, Courtabœuf, France) according to the manufacturer's instructions and stored at -20°C until use. For in vitro cultured testicular explants, the central necrotic area (inherent of long-term culture system) was carefully removed before RNA extraction. To avoid contamination with genomic DNA, extracted RNA was incubated with two units of TURBO DNA-free™ kit (AM1907; Life Technologies™, Carlsbad, California, USA) for 45 min at 37°C. RNA-Seq libraries were generated from 800 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94°C for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.