GSM2263314: TSN3; Homo sapiens; miRNA-Seq
RNA fractions enriched in < 200-nt RNAs were purified from cell pellets using miRNeasy and RNeasy MinElute Cleanup Kits (Qiagen). cDNA libraries were generated using the TruSeq Small RNA library preparation protocol (Illumina). After addition of the TruSeq 3′ adapter (5′ adenylated) using truncated T4 RNA ligase II (New England Biolabs) and, subsequently, the 5′ adapter using T4 RNA ligase I (New England Biolabs), RNA was reverse-transcribed using a 3′ adapter-specific primer. The resulting cDNA was then amplified using 12 cycles of PCR, a universal forward primer and a reverse primer, the latter of which harbors a bar code. cDNA libraries were purified from 8% polyacrylamide and quantified first using an Agilent Bioanalyzer and subsequently using qPCR. Barcoded libraries were mixed at equimolar concentrations and sequenced using a NextSeq 500 machine (Illumina).