GSM8146859: HEK293T, ALAS1-KO, rep3 (miRNA-seq); Homo sapiens; miRNA-Seq
To analyze small RNAs, we isolated biologically independent total RNA samples from HEK293T, ALAS1-KO 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.