GSM7518685: P2631_Sample-TA-62 [CTLX182]; Capra hircus; miRNA-Seq
Total RNAs were extracted from on average of 1 g of powdered of AT and 750 µl of Trizol LS, using a miRVana kit (Thermo Fisher Sciences, USA) following the manufacturer's instruction. The concentration and quality of the RNAs were estimated by spectrophotometry NanodropTH (ND-1000, NanoDrop Technologies LLC, Wilmington, DE, USA) and using the Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA), respectively. Small RNA-Seq librairies were generated from 2 mg of total RNA using TruSeq Small RNA Library Prep Kit (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, during the first step, RNA adapters were sequentially ligated to each end of the RNA; firstly the 3′ RNA adapter (5′ TGGAATTCTCGGGTGCCAAGG 3′) which is specifically designed to target miRNAs and other small RNAs, then the 5′ RNA adapter (5′ GTTCAGAGTTCTACAGTCCGACGATC 3′). Small RNA ligated with 3′ and 5′ RNA adapters were reverse transcribed, and PCR amplified (30 s at 98 °C; [10 s at 98 °C, 30 s at 60 °C, 15 s at 72 °C] × 13 cycles; 10 min at 72 °C) to obtain cDNA. Acrylamide gel purifications of 140–160 nt amplified cDNA (corresponding to cDNA obtained from small RNA + 120 nt from the adapters) were performed. The final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Libraries were loaded in the flow cell at 2.8 nM and clusters were generated using Cbot and sequenced on HiSeq 4000 (Illumina) as single end 50 base reads, according to the manufacturer's instructions.