title: GSM8584532: X3; Bos taurus; miRNA-Seq
library_construction_protocol: Sample collection and preparation RNA qualitative and quantitative analysis (Novogene Experimental Department) RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA,USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the AgilentGVO Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The small RNA libraries were prepared from a total of 2 ug total RNA isolated from each sample using NEBNext Multiplex Small RNA Library Prep Set for Illumina*(NEB, USA.) according to the manufacturer's instruction. Briefly, purified RNA was mixed with NEB 3' SR Adapter, then the SR RT Primer hybridized to the excess of 3'SR Adapter (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adapter into a double-stranded DNA molecule. This step is important to prevent adapter-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase I and therefore do not ligate to the 5'SR Adapter in the subsequent ligation step. 5'ends adapter was ligated to 5'ends of miRNAs, siRNA and piRNA. Then first strand cDNAwas synthesized usingM-MuLV Reverse Transcriptase(RNaseH).PCR amplification was performed using LongAmp Taq2X 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 RNA plus the 3' and 5' adapters) were recovered and dissolved in 8 μL 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 (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on a Novaseq 6000 platform and50 bp single-end reads were generated.
identifiers.namespace: GEO
identifiers.id: GSM8584532_r1