GSM9277870: Ctrl water 2C, biol rep 6; Mus musculus; RNA-Seq
Individual zygotes and 2C embryos were transferred to a drop of M2 medium (Sigma-Aldrich, Merck), then briefly (~10–30 sec) held in a drop of Tyrode's Solution, Acidic (Sigma-Aldrich, Merck) to remove the zona pellucida. Next, the zygote/2C embryo was swiftly washed through three drops of DPBS (without calcium and magnesium chloride). In an RNase-free environment, the zygote/embryo was carefully pipetted together with 1 µl DPBS into an 8-tube 0.2 ml PCR strip (Axygen) containing 6 µl DPBS supplemented with RiboLock RNase inhibitor (1:200; Thermo Fisher Scientific) in each tube. Once the strip was filled, samples were flash frozen by submerging the bottom part of the tubes in LN2 and then transferred to -80 °C for storage until library preparation. Library preparation was performed with SMART-Seq Stranded Kit (634443, Takara Bio) for full transcriptome analysis. Libraries of zygotes and 2C embryos were prepared on two separate days following the kit's user manual starting from intact cells (option 1, with fragmentation). For PCR1, we used indexes from a SMARTer RNA Unique Dual Index Kit – 96U Set A (634452, Takara Bio) to avoid index hopping, and we used 10 cycles (ultra-low input workflow). We did not pool the samples after PCR1. For PCR2, we used 13 cycles, based on a prior pilot experiment. Final RNA-seq libraries were purified using AMPure beads (Agencourt AMPure XP, Beckman Coulter) with a single round of clean-up to minimise sample loss. Successful removal of adapter dimers and appropriate library size distribution were confirmed using an 2100 Bioanalyzer (Agilent). Libraries were quantified with Qubit dsDNA HS kit (Thermo Fisher Scientific), pooled in equimolar ratios, and subjected to a final 1.2x AMPure bead purification. The pooled library was eluted in EB buffer (10 mM Tris-HCl, pH 8.5) and submitted for sequencing. Sequencing was performed at the Norwegian Sequencing Centre (Department of Medical Genetics Ullevål, Oslo University Hospital) on an Illumina NovaSeq 6000 using an SP flow cell with 200 cycles, generating 100 bp paired-end reads.