GSM9635077: SV40-MES-13, WT, miRNAseq, rep2; Mus musculus; miRNA-Seq
Total RNA was extracted from the mouse renal mesangial cell line ME13 sv40 (both DROSHA knockdown and control groups) using a standard phenol-chloroform-based method (TRIzol reagent). The protocol involved cell lysis, phase separation with chloroform, RNA precipitation with isopropanol, and washing with ethanol. To specifically enrich for small RNA species (including microRNAs), the total RNA fraction was further processed. Small RNAs (approximately 18-30 nucleotides) were size-selected and purified using a commercial kit (mirVana™ miRNA Isolation Kit or similar) according to the manufacturer's instructions. The final RNA concentration and integrity were assessed using a spectrophotometer (NanoDrop) and a bioanalyzer ( Agilent 2100 Bioanalyzer with the Small RNA Kit), respectively, to ensure high-quality input material for library construction. Small RNA sequencing libraries were constructed from the enriched small RNA samples using a dedicated commercial kit(NEBNext® Multiplex Small RNA Library Prep Set for Illumina). The protocol typically involved the following key steps: First, 3' and 5' RNA adapters were sequentially ligated to the small RNAs. The adapter-ligated RNA was then reverse-transcribed into cDNA. Following this, the cDNA was amplified via PCR using primers containing unique index sequences to allow for sample multiplexing. The final PCR products, representing the small RNA library, were size-selected to isolate fragments corresponding to the desired library size range (typically ~140-160 bp for mature microRNAs) using gel electrophoresis or magnetic bead-based purification. Library quality and concentration were validated using a High Sensitivity DNA assay on a bioanalyzer or by qPCR, and were then pooled in equimolar amounts for sequencing on an NextSeq  Illumina platform.