GSM6208766: wildtype 49/52 EN1+ fibroblasts; Mus musculus; RNA-Seq
Based on the amount of cells homogenized in the RLT buffer (79216, Qiagen), the total RNA was isolated using the RNeasy Mini Kit (74106, Qiagen) and RNeasy Micro Kit (74004, Qiagen), including an on column DNase digestion (79254, Qiagen), according to the manufacturer's instructions. Quality and quantity of the total RNA was assessed on the 2100 Bioanalyzer instrument following manufacturer's instructions “Agilent RNA 6000 Nano” (G2938-90034, Agilent Technologies) and “Agilent RNA 6000 Pico” (G2938-90046, Agilent Technologies). Total RNA samples having RIN>7 were subjected to TruSeq stranded mRNA library preparation, according to the manufacturer's instructions (Document # 1000000040498 v00 , Illumina). The stranded mRNA libraries were analyzed on a 2100 Bioanalyzer instrument following the manufacturer's protocol “Agilent DNA 7500 kit” (G2938-90024, Agilent Technologies), diluted to 10nM and pooled equimolar into multiplex sequencing pools for sequencing on the HiSeq 2500 and NovaSeq 6000 Illumina sequencing platforms. NovaSeq 6000 paired-end sequencing was performed using 54 cycles for Read 1, 19 cycles for Read i7, 10 cycles for Read i5 and 54 cycles for Read 2, using the NovaSeq6000 SP Reagent Kit v1.5 (100 cycles) (20028401, Illumina). RNA libraries were prepared for sequencing using standard Illumina protocols