GSM7728333: Sperm_LPD_Male11 [LPD11]; Mus musculus; RNA-Seq
RNA was extracted from total sperm (approx. 4x10^6 to 8x10^6 cells per sample) using miRNeasy micro Qiagen kit with Qiazol homogenisation on TissueLyser. Stranded RNA-seq libraries were prepared 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). Prior to library preparation, total RNA was treated with QIAseq FastSelect -rRNA HMR, to prevent any rRNA present from being converted into sequencing library. 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.