miRNA  profiling analysis of pig induced pluripotent stem cells by high-throughput sequencing
miRNA  profiling analysis of pig induced pluripotent stem cells by high-throughput sequencing
The piPS cells were maintained on mitotically inactive mouse embryonic fibroblasts (MEFs) feeder derived from ICR mice supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, beta-mercaptoethanol, and LIF, bFGF. Total RNA was isolated from cell cultures or snap-frozen tissues, in biological triplicates or greater, using TRIzol extraction and purified with the Qiagen RNA Purification Kit. A total amount of 3 μg 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, USA.) following manufacturer’s recommendations and index odes were added to attribute sequences to each sample. Briefly, NEB 3-prime SR Adaptor was directly and specifically ligated to 3-prime end of miRNA, siRNA and piRNA. After the 3-prime ligation reaction, the SR RT Primer hybridized to the excess of 3-prime SR Adaptor (that remained free after the 3-prime 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 prime SR Adaptor in the subsequent ligation step. 5 prime ends adapter was ligated to 5 prime 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-prime and 5-prime adaptors) were recovered and dissolved in 8 uL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturers instructions
Experimental Factor: compound
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