GSM8229144: MDWD1; Mus musculus; RNA-Seq
Total RNA was isolated from snap-frozen (liquid nitrogen) samples of maternal liver at late gestation using the RNeasy Mini plus kit (Qiagen, UK), in accordance with the manufacturer's instructions. RNA was treated with QIAseq FastSelect -rRNA HMR, to prevent any rRNA present from being converted into the sequencing library Stranded RNA-seq libraries were prepared from 500 ng of total RNA per sample, using the NEBNext Ultra II Directional RNA Library Preparation Kit for Illumina (NEB; E7760) and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Pairs) (NEBNext; E6440). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific; Q32854). Library fragment-length distributions were analysed using the Agilent 4200 TapeStation and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584 and 5067-5585). Libraries were pooled in equimolar amounts and final library quantification was performed using the KAPA Library Quantification Kit for Illumina (Roche; KK4824). The library pool was sequenced on the Illlumina NextSeq500 over three NextSeq500 High Output 150 cycle kits (Illumina; 20024907), to generate over 40 million pairs of 75-bp paired-end reads per sample.