Name: GSM7842498
Instrument: NextSeq 550
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: cDNA
Layout: PAIRED
Construction protocol: MII oocytes and 2-cell embryos were collected as described before. For 2-cell embryos, the zona pellucida was removed with Tyrode's solution (SIGMA-ALDRICH), the blastomeres were separated and the polar body was discarded. Then single oocytes and blastomeres were picked and put into a lysis buffer (Buffer RLT Plus Qiagen). DNA and RNA from lysate were separated using the G&T protocol (Angermueller et al. 2016). The magnetic beads (MyOne C1, Life Technologies) were washed and then annealed to oligo dTs. These were used to capture polyadenylated mRNA from individual cell lysate. The supernatant containing the DNA was transferred to a new 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 of RNAsin (Promega) to remove all DNA residue. Each washing solution was added to the DNA tube to maximise recovery. mRNA on the beads was processed further for cDNA conversion. This involved the resuspension in 10 μl of reverse transcriptase mastermix (100 U SuperScript II (Invitrogen), 10 U RNAsin (Promega), 1 × Superscript II First-Strand Buffer, 5 mM DTT (Invitrogen), 1 M betaine (Sigma), 9 mM MgCl2 (Invitrogen), 1 μM Template-Switching Oligo (TSO, Eurogentec), 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 obtained was subjected to PCR amplification by adding 11 μl of 2x KAPA HiFi HotStart ReadyMix and 1 μl of ISPCR primer (2 μM). The amplification was carried out in a thermocycler at 98 °C for 3 min, followed by 15 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and the final extension step at 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. 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. All 96 single-cell RNA-seq libraries were pooled together and sequenced on the Illumina NextSeq platform to an average depth of 4.2 million reads, using paired-end 75 bp read-length settings.