GSM8459435: miRNA hESCs, Dicer-S1344L hemizygous, Rep1; Homo sapiens; miRNA-Seq
To analyze small RNAs, we isolated biologically independent total RNA samples from hESC cells using Trizol (Invitrogen) One milligram of total RNA was used to generate small RNA libraries for each sample. After adding 1 μL of QIAseq TM miRNA Library QC Spike-Ins (QIAGEN) to the total RNA, a 3' linker was ligated to it in a reaction containing 100 mM 3' linker, T4 RNA Ligase 2, truncated KQ (NEB), 10% PEG8000, 1x RNA ligation buffer, and 20 U RNase OUT overnight at 17°C. The 48-57 nucleotide sized 3' linker-ligated small RNAs were purified using 12% Urea-PAGE gel with radio-labeled oligonucleotides serving as size-markers. Subsequently, the small RNA-linker hybrid was subjected to a 5' ligation reaction overnight at 17°C containing 100 mM 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 then reverse transcribed, PCR amplified using 12 cycles of PCR with forward and Illumina index reverse primers, and purified by 8% non-denaturing acrylamide gel. Sequencing was performed with HiSeq 4000 in SE50 mode.