title: GSM4484104: P0I-405251-T-RNA [S117]; Mus musculus; ssRNA-seq
library_construction_protocol: Tumor specimens and matched liver control were harvested and stored in RNALater at -80° until all samples were collected. DNA and RNA extractions from each sample were performed using AllPrep DNA/RNA Mini Kit (Qiagen). Extracted total RNA quality and concentration was assessed on a 2100 Bioanalyzer (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific), respectively. Only extracts with RNA Integrity Number (RIN) greater than 7 were processed for sequencing. DNA extraction was followed by RNAse treatment (Qiagen). Genomic DNA samples were quantified using fluorometric quantitation on the Qubit 2.0 (ThermoFisher Scientific). For each sample, 200 ng of DNA was sheared using focused-ultrasonicators (Covaris) to generate DNA fragments of about 300 bp in length. RNA-seq libraries were prepared using the commercially available KAPA Stranded mRNA-Seq Kit (Roche) following the manufacturer's protocol. In brief, mRNA transcripts were first captured using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed using random priming. During the 2nd strand synthesis, the cDNA:RNA hybrid was converted into to double-stranded cDNA (dscDNA) and dUTP incorporated into the 2nd cDNA strand, effectively marking the second strand. Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. The strand marked with dUTP was not amplified, allowing strand-specificity sequencing. Libraries were indexed using a dual indexing approach allowing for multiple libraries to be pooled and sequenced on the same sequencing Illumina sequencing flow cell.
attributes.tag: GEO Accession
attributes.value: GSM4484104