GSM5155388: LDG; Ovis aries; ncRNA-Seq
Peripheral blood mononuclear cells (pbmc) were separated from blood samples taken from lambs and RNA was isolated with TRI Reagent (Sigma). miRNA libraries (LDG and HDG groups) were prepared with NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) with 3 ug of total RNA. NEB 3' SR Adaptor was directly and specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adaptor into a double stranded DNA molecule. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–). 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 corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and 50bp.