GSM8412826: Normal-4 tsRNA; Homo sapiens; RNA-Seq
Deparaffinization of paraffin-embedded FFPE tissue samples and total RNA was extracted with TRIzol reagent (#15596026, Invitrogen). Sample integrity and concentration were examined by agarose gel electrophoresis and NanoDrop One (Thermo Fisher Scientific, MA, USA). Using the rtStar™ tRF&tiRNA Pretreatment Kit (#AS-FS-005, Arraystar) to remove the 3'-carbamoyl and 3'-cP termini, phosphorylation of the 5'-OH terminus, and demethylation of m1A, m1G, and m3C demethylation to prepare RNA samples suitable for tsRNAs sequencing library construction [29]. The extracted total RNA samples were ligated one by one with small RNA junctions at the 3' and 5' ends, and the cDNA was synthesized and amplified using proprietary reverse transcription primers and amplification primers (Illumina, CA, USA). Approximately 134-160 bp of PCR amplified fragments were extracted and purified from PAGE gels to ensure the purity and quality of the final PCR amplified fragments obtained. The concentration and purity of the tsRNAs library was determined using an Agilent 2100 Bioanalyzer (Agilent, CA, USA). The library was denatured and diluted to a volume of 1.3 mL at a concentration of 1.8 pM.