GSM8865954: CMT93 spheroids treated with vehicle, replicate 1; Mus musculus; RNA-Seq
For RNA extraction, spheroids were collected, centrifuged, and washed once with PBS. Cell pellets were then lysed with TRIZOL reagent, and total RNA was isolated according to the manufacturer's instructions. RNA concentration and purity were determined spectrophotometrically using a Nanodrop ND-8000, and RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 Eukaryote Total RNA Nano Assay. 1000 ng of total RNA per sample were used as input for library preparation. Using the Illumina Stranded mRNA Sample Prep Kit (protocol version: # 1000000124518 v02 (April 2021)), poly-A containing mRNA molecules were purified from the total RNA input using poly-dT oligo-attached magnetic beads. The purified mRNA was fragmented and in a reverse transcription reaction using random primers and Actinomycin D, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using dUTP to achieve strand specificity. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which pre-index anchors were ligated preparing the fragments for dual indexing. Anchor-ligated fragments were then purified using magnetic beads. Finally, enrichment PCR was carried out to enrich those DNA fragments that have anchor-ligated DNA fragments and to add indexes and primer sequences for cluster generation.
GEO
GSM8865954_r1