GSM6245774: TUT247_rep2; Homo sapiens; miRNA-Seq
We extracted RNA from HEK293T cells using Trizol (Invitrogen) according to the manufacturer's instructions. 1 ug of total RNA were used to generate small RNA libraries for each sample. 3' linker was ligated to total RNA in the reaction containing 100μM 3' linker, T4 RNA Ligase 2, truncated KQ (NEB), 10% PEG8000, 1X RNA ligation buffer, and 20 U RNase OUT overnight at 17°C. Using radio-labeled oligonucleotides as size-markers, 48-57 nucleotide sized 3' linker-ligated small RNAs were purified by 12% Urea-PAGE gel. The small RNA-linker hybrid was then subjected to 5' ligation reaction overnight at 17°C containing 100 μM 5' adaptor, 20% PEG8000, 1X RNA ligase buffer, 1 mM ATP, 20 U RNaseOUT and 5 U T4 RNA ligase 1. The linker ligated RNAs were reverse transcribed, and PCR amplified using 12 cycles of PCR with forward and Illumina index reverse primers and the amplified libraries were purified by 8% non-denaturing acrylamide gel.