GSM5221843: C3_S3_R1_001; Homo sapiens; miRNA-Seq
Total RNA was extracted using TRIZOL reagent (Invitrogen). RNA concentration and quality were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), which calculates an RNA integrity number (RIN). Total RNA with A260/A280 = 1.8-2.0 and RIN >8.0 were selected. Intact RNA samples were normalized to 200 ng/μl with DEPC-treated H2O. Total 2μg RNA was used in microRNA library preparation. The first step of microRNA library preparation involves 3'end ligation using a 5' adenylated DNA adapter with a blocked 3'end. Using a truncated version of T4 RNA Ligase 2, the ATP-less reaction is driven by the adenylated group on the adapter, resulting in ligation to molecules with a 3'hydroxy group.  The second step of microRNA library preparation involves a 5'RNA adapter which is ligated to the 5'phosphate end of microRNA by T4 RNA Ligase 1. RNA molecules with a 5'phosphate are the only substrates for this ligation reaction.  The third step of microRNA library preparation involves annealing a reverse transcription primer to the 3'ligated adapter and extension. Then followed by PCR amplification using a pair of primers that add on Illumina flow cell binding sequences and barcodes. The final amplified small RNA library consists of approximately 120 bases of adapter sequence plus an insert of between 22 - 30 bases. Higher molecular weight products are removed by PAGE purification.  Single-read 75 nucleotide reads from each microRNA library were obtained using NextSeq500 (Illumina Inc., CIC bioGUNE, Bilbao, Spain).