GSM3535472: I_2; Homo sapiens; miRNA-Seq
Total RNA was extracted from the frozen hippocampal tissues using Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity and concentration were assessed using the NanoPhotometer® spectrophotometer (Implen GmbH, Munich, Germany) and the Qubit® RNA Assay Kit in the Qubit® 2.0 Flurometer (Thermo Fisher Scientific). The RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) was used to measure RNA integrity. 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 codes were added to attribute sequences to each sample.Briefly, 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, dsDNAsare not substrates for ligation mediated by T4 RNA Ligase 1 and therefore donot 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 140~160bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessedon the Agilent Bioanalyzer 2100 systemusing DNA High Sensitivity Chips.
GEO Accession
GSM3535472