GSM4838956: Specimen-N2; Homo sapiens; ncRNA-Seq
Total RNAs were extracted according to the manufacturer's instruction (Invitrogen, CA,USA). The purity and concentration of total RNA samples were determined with NanoDrop ND-1000. Then we did the following treatments to remove some RNA modifications which might interfere with small RNA-sequencing library construction: 3'-aminoacyl (charged) deacylation to 3'-OH (hydroxyl group) for 3' adaptor ligation, 3'-cP (2',3'-cyclic phosphate) removal to 3'-OH for 3' adaptor ligation, 5'-OH phosphorylation to 5'-P for 5'-adaptor ligation, N1-methyladenosine and N3-methylcytidine demethylation for efficient reverse transcription. All procedures were performed using rtStar tRF&tiRNA Pretreatment Kit (Arraystar) protocols. Total RNA samples were sequentially ligated to 3' and 5' small RNA adapters using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA). cDNA was then synthesized and amplified using Illumina's proprietary reverse transcription primers and amplification primers. Subsequently, 134-160 bp PCR amplified fragments (corresponding to 14-40 nt small RNA size range) were extracted and purified from the PAGE gel. The completed libraries were quantified by Agilent 2100 Bioanalyzer. Libraries were mixed in equal amounts according to the quantification results and used for sequencing on the instrument. And then, the DNA fragments in well-mixed libraries were denatured to generate single-stranded DNA molecules and loaded onto the reagent cartridge. Finally, the sequencing run was performed on Illumina NextSeq 500 system using NextSeq 500/550 V2 kit (Illumina) according to the manufacturer's instructions. Sequencing was carried out by running 50 cycles.