GSM8197231: B6-Ctrl; Mus musculus; RNA-Seq
Organoids were collected by centrifugation and RNA was extracted using Trizol The NEBNext Ultra II directional RNA library prep kit for Illumina (New Englands Biolabs Inc., Ipswich, MA, USA) was used to prepare mRNA sequencing libraries, according to manufacturer's instruction. Briefly, 400 ng of total RNA were purified using the NEBNext poly(A) mRNA Magnetic Isolation module (New Englands Biolabs Inc., Ipswich, MA, USA) and 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 10 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 QuBit 3.0 fluorometer (ThermoFisher Scientific, Canada). Subsequently, mRNA-seq librairies with unique dual index were pooled together in equimolar ratio and sequenced for paired-end 100 pb sequencing on an Illumina NovaSeq 6000 at the Next-Generation Sequencing Platform, Genomics Center, CHU de Québec-Université Laval Research Center, Québec City, Canada. The mean coverage/sample was 20M paired-end reads.