title: GSM8993728: RNA-seq of mouse ovary tissue (OT1); Mus musculus; RNA-Seq
library_name: GSM8993728
library_construction_protocol: RNA library was prepared using an Illumina TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, after removing rRNA from the total RNA (700 ng) using an rRNA removal kit, RNA was purified using beads. After rRNA depletion, a fragment mix (EPH) was used for the remaining RNA at 94°C for 6 min. Fragmented RNA primed with random hexamers was transcribed to first strand cDNA using reverse transcriptase and random primers at: 25°C for 10 min, 42°C for 15 min, and 70°C for 15 min. to produce double stranded cDNA, the replacement strand was synthesized by incorporating dUTP in place of dTTP using polymerase at 16°C for 1 h. After cDNA was cleaned using sample purification beads, a single “A” nucleotide was attached to the 3' ends of the blunt fragments using A-Tailing mix reagent by incubation at 37°C for 30 min, and then at 70°C for 5 min. Indexing adapters were ligated to the ends of the DNA fragments using a ligation mix 2 reagents at 30 °C for 10 min. PCR was conducted to enrich the DNA fragments with adapter molecules on both ends after washing twice with sample purification beads under the thermocycler conditions as follows: 95°C for 3 min, followed by eight cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 30 min, with a final extension at 72°C for 5 min.