GSM3855769: WT Ctrl 2; Mus musculus; RNA-Seq
Trizol extraction of total RNA was performed according to the manufacturer's instructions We used the Illumina NextSeq 500 and the latest versions of sequencing reagents and flow cells. Total RNA was subjected to 2 rounds of poly(A)+ selection and converted to cDNA. We used TruSeq library generation kits, as per the manufacturer's instructions (Illumina, San Diego, California). Library construction consisted of random fragmentation of the poly(A) mRNA, followed by cDNA production using random primers. The ends of the cDNA were repaired and A-tailed, and adaptors were ligated for indexing (with up to 12 different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit (Kapa Biosystems, Woburn, Massachusetts) prior to cluster generation. Clusters were generated to yield approximately 725K–825K clusters/mm2. Cluster density and quality was determined during the run after the first base addition parameters were assessed. We ran paired-end 2 × 75–bp sequencing runs to align the cDNA sequences to the reference genome