GSM9726425: BW_1_miRNA; Rattus norvegicus; miRNA-Seq
Total RNA enriched for small RNAs was extracted from 200 µL of sample using QIAzol Lysis Reagent. After adding chloroform and centrifuging at 12,000×g for 15 minutes at 4°C, the aqueous phase was collected and mixed with 1.5 volumes of ethanol. The mixture was applied to a spin column, washed with RWT and RPE buffers, and miRNA was finally eluted in 30 µL RNase-free water. Total RNA was ligated with 3' and 5' adapters sequentially using T4 RNA ligases. Reverse transcription was performed with RT primer complementary to the 3' adapter, followed by PCR amplification (15–18 cycles) using indexed primers. The amplified cDNA libraries were size-selected on a 6% Novex TBE gel (140–160 bp), purified, and quantified by Qubit before sequencing on Illumina NovaSeq 6000.