GSM8447521: D-13_SN_scRNA-seq; Mus musculus; RNA-Seq
Fully-grown germinal vesicle (GV) oocytes were mechanically dissected from the ovaries of two 12-week-old C57Bl6/Babr mice in M2 medium (Sigma-Aldrich, M7167). After removing all cumulus cells, oocytes were placed in M2 with Hoechst (5µM, Abcam, 33343) and incubated for 10 minutes at room temperature. Oocytes were washed in PBS and NSN/SN stage was scored on a Zeiss LSM780 confocal microscope. Absence of a ring around the nucleolus was counted as “NSN”, a partial ring as “intermediate” and a full ring “SN”. After scoring, oocytes were frozen individually in 5μl RLT Plus buffer (Qiagen) and stored at -80°C until further use. DNA and polyadenylated mRNA from individual oocytes were physically separated using poly-dT bound magnetic beads according to the G&T protocol published by Angermueller et al. 2016.  Bead-bound mRNA was transcribed into cDNA using SuperScript II (Invitrogen) and Template-Switching Oligo primers (TSO, Eurogentec), followed by amplification of cDNA with the 2x KAPA HiFi HotStart ReadyMix and ISPCR primers (Picelli et. al, 2013, Picelli et. al, 2014). Libraries were prepared from 100 to 400 pg of cDNA using the Nextera XT Kit (Illumina), per the manufacturer's instructions but with one-fifth volumes.