title: GSM4307105: WT5 I; Mus musculus; RNA-Seq
library_construction_protocol: The NEBNext Ultra II directional RNA library prep kit for Illumina (New Englands Biolabs Inc., Ipswich, MA, USA) was used to prepare total RNA sequencing libraries, according to manufacturer's instruction. Briefly, 100 ng of total RNA was used. Ribosomal RNA (rRNA) including both cytoplasmic and mitochondrial rRNA was removed using RNaseH-based method (NEBNext rRNA depletion kit; New Englands Biolabs Inc., Ipswich, MA, USA). Following purification with Agencourt RNAClean XP beads (Beckman Coutler, Missisauga, Ontario, Canada), the RNA was fragmented using divalent cations under elevated temperature. The fragmented RNA was used as a template for cDNA synthesis by reverse transcriptase with random primers. The specificity of the strand was obtained by replacing the dTTP with the dUTP. This cDNA was subsequently converted to double-stranded DNA and end-repaired. Ligation of adaptors was followed by a purification step with AxyPrep Mag PCR Clean-up kit (Axygen, Big Flats, NY, USA), by an excision of the strands containing the dUTPs and finally, by a PCR enrichment step of 11 cycles to incorporate specific indexed adapters for the multiplexing. The quality of final amplified libraries was examined with a DNA screentape D1000 on a TapeStation 2200 and the quantification was done on the QBit 3.0 fluorometer (ThermoFisher Scientific, Canada). Subsequently, total RNA-seq librairies with unique index were pooled together in equimolar ratio and sequenced for paired-end 125 pb sequencing using one lane of a high ouput flowcell on an HiSeq 2500 V4 system at the Next-Generation Sequencing Plateform, Genomics Center, CHU de Québec-Université Laval Research Center, Québec City, Canada.