title: GSM4787724: HCT116 cells transfected with pCDNA3.0 pri-mir-342midB_none plasmid replicate 3; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNAs were extracted using TRIzol reagent. 1.5 μg of pcDNA3.0 plasmids encoding pri-mir-342 or its variants plus 0.5 μg of pcDNA3.0 encoding pri-mir-1266 were co-transfected into HCT116 cells pre-seeded in one well of 6-well plate using lipofectamine 3000 (Thermo Fisher Scientific). One and a half days after transfection, total RNAs were extracted from the transfected cells using TRIzol reagent (Invitrogen). Fifteen μg of each total RNA was loaded into 15% urea-PAGE. The small RNAs within the 19-24 nt region were gel-purified. The purified small RNAs were ligated to the 3'-adapter (4N-RA3) using T4 RNA ligase 2, truncated KQ (NEB). The 3'-adapter ligated RNAs were separated from free 3'-adapter and unligated small RNAs by gel-purification. After which, the purified 3'-adapter RNAs were ligated with 5'-adapter (RA5-4N) using and T4 RNA ligase 1 (NEB). Next, the 3'- and 5'-adapter-ligated RNAs were reverse transcribed using SuperScript™ IV Reverse Transcriptase with the R-RA3 primer, which generated cDNAs. The resulting cDNAs were amplified using PCR with RP1 and RPI primer (Illumina), which produced DNA libraries. We used a RPI primer with a unique index sequence for each small RNA sample.