GSM4669761: yGV_2 RNA-seq; Mus musculus; RNA-Seq
Oocytes were released from the ovaries mechanically with a fine needle and cleaned in M2 medium (Sigma). Clean oocytes were washed twice in PBS, inspected to ensure complete removal of adherent granulosa cells, then individually lysed and flash-frozen in 5μl RLT Plus buffer (Qiagen) and stored at -80°C until further use. DNA and RNA from individual oocytes were physically separated using the G&T protocol (Angermueller et al., 2016). Briefly, Smart-seq2 oligo-dTs (Picelli et al., 2013, 2014) were annealed to magnetic beads (MyOne C1, Life Technologies) and used to capture polyadenylated mRNA from individual oocyte lysates. The remaining lysate containing the DNA was transferred to a separate tube and the beads washed three times in 1xFSS buffer (Superscript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma) and 0.4 U μl−1 of RNAsin (Promega). The washing solutions were added to the DNA tube to maximise recovery. mRNA on the beads was immediately processed further for cDNA conversion by resuspending beads in 10 μl of reverse transcriptase mastermix (100 U SuperScript II (Invitrogen), 10 U RNAsin (Promega), 1 × Superscript II First-Strand Buffer, 2.5 mM DTT (Invitrogen), 1 M betaine (Sigma), 9 mM MgCl2 (Invitrogen), 1 μM Template-Switching Oligo (TSO, Exiqon (Picelli et al., 2013, 2014)), 1 mM dNTP mix (Roche)). The mRNA mixture was reverse transcribed by incubation for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C. The cDNA was amplified by PCR by adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl ISPCR primer (2 μM; (Picelli et al., 2013, 2014)). Samples were incubated in a thermocycler at 98 °C for 3 min, followed by 18 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. The amplified product was purified using Ampure XP beads with a 1:1 ratio and eluted into 20 μl of water. In parallel, the lysate containing the DNA was purified using a 0.8:1 volumetric ratio of Ampure XP Beads (Beckman Coulter) and eluted into 10 μl of water to use for scBS-seq library preparation. 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.
GEO Accession
GSM4669761