GSM5013469: Quiescent HASMCs rep3; Homo sapiens; ncRNA-Seq
RNAsimple Total RNA Kit was used to extract total RNA from proliferative and quiescent HASMCs. In order to eliminate the interference of RNA modification on the construction of small RNA-seq library, the total RNA samples were first pretreated as follows: 3ʹ-aminoacyl (charged) was deacylated to 3ʹ-OH for 3ʹ adaptor ligation, 3ʹ-cP (2ʹ, 3ʹ-cyclic phosphate) was removed to 3ʹ-OH for 3ʹ adaptor ligation, 5ʹ-OH (hydroxyl group) was phosphorylated to 5ʹ-P for 5ʹ-adaptor ligation, m1A and m3C were demethylated for effective reverse transcription. Subsequently, the commercial kit for tRF & tiRNA sequencing library preparation was used to construct the tsRNA-seq libraries. The kit includes 3ʹ-adapter and 5ʹ-adapter ligation adaptor ligation, cDNA synthesis and library PCR amplification. PCR amplified fragments with a size of 135~160 bp (corresponding to the size range of 15~40 nt small RNA) were selected as tsRNA-seq libraries. Finally, the prepared tsRNA-seq libraries were quantified using Agilent BioAnalyzer 2100, and then sequenced using Illumina NextSeq 500.