GSM8834858: TSq1; Homo sapiens; RNA-Seq
Total RNA was isolated using the mirVanaTM miRNA iolation kit (Ambion) with minor modifications. 350μl of TriReagent was used for cell lysis and storage of samples, followed by addition of 350μl of mirVana cell lysis solution and 35μl of mirVana miRNA additive. All further steps for the isolation of total RNA were performed as recommended by the manufacturer. Quality and quantity of isolated RNA was assessed by agarose gel electrophoresis and spectrophotometry (Nanodrop) respectively. For library preparation, the QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD with UDI (Lexogen) was used according to manufacturer's instructions. Briefly, 150-400 ng of RNA from each sample were used for 1 st strand synthesis, followed by RNA template removal. 2 nd strand synthesis was initiated by random primers containing compatible linker sequences at their 5' end. In-line barcodes were introduced during second-strand synthesis, followed by magnetic bead-based purification. The resulting products were amplified for up to 15 cycles and re-purified. Library quality was assessed using the Agilent Tapestation 4150 and the High Sensitivity D1000 assay kit, while quantity was assessed using the Invitrogen Qubit 4 Fluorometer and the Invitrogen Qubit dsDNA HS Assay. The quantified libraries were pooled equimolarly, and a total of 25ng were used for Adapter Conversion PCR Amplification using the Universal Library Conversion Kit (App-A), Version: V1.0 (MGI Tech Co., Ltd.), following the Universal Library Conversion Kit (App-A) User Manual (Version: A3).
GEO
GSM8834858_r1