Goat skeletal muscle F45(1)
Total RNA isolation using TRIzol reagent (Invitrogen, Carlsbad, CA) on 21 longissimus dorsi muscle samples. Then, RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 3 g total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, Ipswich, MA, USA) and index codes were added to attribute sequences to each sample, followed by reverse transcription. PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for illumina and index (X) primer. PCR products were purified on a 8% polyacrylamide gel (100V, 80 min). DNA fragments of 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 L elution buffer. And library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. Finally, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) and the library preparations were sequenced on an Illumina Hiseq 2500 platform and 50bp single-end reads were generated.