GSM6570927: pooled oocyte null-3; Mus musculus; RNA-Seq
COCs were collected from the ampulla of the oviduct. Denuded oocytes were prepared by treating COCs with 3 mg/ml hyaluronidase for 2 minutes at room temperature. Oocytes from 3-4 mice of the same genotype were pooled to obtain pools of approximately 150 oocytes in each sequenced RNA sample. RNA was isolated from four Nlrp2-null and three WT oocyte pools using the Qiagen miRNeasy kit (QIAGEN, US) and used for the preparation of RNA-Seq libraries. Libraries were prepared with the NuGEN RNA-Seq library preparation protocol. Briefly, purified double-stranded cDNA was generated from approximately 20 ng of total RNA and amplified using both oligo d(T) and random primers. Five hundred nanograms of each sample's ds-cDNA were sheared using the Covaris S2 focused-ultrasonicator to a target size of 400 bp. A double-stranded DNA library was then generated from 1 g of sheared, double-stranded cDNA, preparing the fragments for hybridization to the flowcell. Using the concentration from the ViiA7TM qPCR machine, 14 pM of the equimolarly pooled library is loaded onto an Illumina HiSeq 2500 sequencing instrument (Illumina, USA).