GSM5210485: Embryo rep6; Mus musculus; RNA-Seq
Samples were collected in TRIzol (Thermo Fisher) and kept at -80 °C until further processed. RNA was prepared following the manufacturer's protocol with the following modifications: The aqueous solution was precipitated overnight using isopropanol and 10 μg of glycogen (Ambion) to increase the recovery of RNA. Samples were centrifugated at 12,000 g for 45 min, washed twice with 70% EtOH and RNA was dissolved in RNAse-free water (Qiagen). Samples were quantified using QuBit fluorometers (Thermo Fisher) and RNA purity was measured using a Nanodrop. A Bioanalyser RNA 6000 Pico assay (Agilent) was used to check the RNA quality. RNA samples with an RNA integrity number (RIN) above 9 (9–9.5) were selected and sent to the Oxford Genomic Centre (Oxford, UK) for the RNAseq experiment. Samples were normalised to a 50 ng input. The purification of mRNA, generation of double-stranded cDNA and library construction were performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with the centre's adapters and barcode tags, before sequencing on an Illumina HiSeq 4000 as 75 bp paired end.