GSM5628254: Liver, treated Al/NP high - rep3; Mus musculus; RNA-Seq
Total RNA was extracted from fresh frozen pieces of mouse liver using Qiagens RNeasy Mini Kit. Quantity and quality was assessed using the Qubit 3 fluorometer with the RNA BR AssayKit (Invitrogen) and the Agilent Bioanalyzer 2100 system using the RNA Pico kit. mRNA-focused RNA libraries were prepared using Illumina's TruSeq stranded RNA library prep kit with an input amount of 1000ng total RNA. Poly(A)-positive mRNA transcripts were isolated from total RNA by binding to magnetic oligo(d)T beads and subsequently fragmented and reverse transcribed during first and second strand synthesis to yield double-stranded cDNA. The ends of the cDNA molecules were end repaired and an adenosine overhang was added to all 3' ends (A-tailing step) to facilitate the ligation of the adapter molecules. These adapters have individual dedicated index sequences and add the Illumina specific sequences that are needed for amplification, flow cell hybridization and sequencing. 8 bp single-index NEXTflex DNA barcodes (Perkin Elmer) were used. The final ligation product was amplified via PCR. Intermediate and final library purification steps were done using AMPure XP beads (Beckman Coulter). Final library quantity and quality was done using the Qubit 3 fluorometer with the dsDNA HS AssayKit (Invitrogen) and the Agilent Bioanalyzer 2100 system using the High Sensitivity DNA kit. All mRNA libraries were sequenced in paired-end mode (2 x 50 nt) on an Illumina NovaSeq 6000 instrument resulting in around 30 million distinct sequencing read pairs per library.