GSM5662926: N14_SMT2_MII_KO_sc18; Mus musculus; RNA-Seq
To collect preimplantation embryos, 6-8week old wild-type and Nlrp14-/- females (C57BL/6) were super-ovulated by injection of PMSG and hCG. MII oocytes were collected at 14-15 hours after hCG injection. As to preimplantation embryos, superovulated female mice were mated with either C57BL/6 or PWK males. Embryos were collected at 25-26 h (zygotes), 42-43 h (2-cell), 56-57 h (4-cell), 70-71 h (8-cell), and 94-96 h (blastocysts) after hCG injection. To isolate pronuclei, mouse zygotes at PN3- PN4 stages were collected and pronucleus were isolated as previously described (Guo et al., 2014). Zygotes which were maternally deficient in Tet3 were obtained through Zp3-Cre mediated conditional knockout in oocytes as previously described (Guo et al., 2014). To inhibit DNA replication, zygotes were treated with aphidicolin until the collection of pronuclei (Guo et al., 2014). The inhibition of DNA replication in mouse zygotes was validated by EdU incorporation assay. ACE-seq: The construction of ACE-seq libraries followed the published protocol (Schutsky et al., 2018), with minor modifications. Fragmented gDNA was first modified using UDP- glucose and T4 Phage β-glucosyltransferase at 37 °C for 1 h. Then 0.1 N NaOH was added to the modified gDNA for denaturation at 55 °C for 10 min. APOBEC enzyme was used to deaminate gDNA at 37°C for 3 hrs. After deamination, gDNA was purified using Ampure XP beads before library construction according to the TAILS procedure (Gu et al., 2019; Yan et al., 2021). Finally, the ACE-seq libraries were sequenced on a Novaseq sequencer for 150 bp paired-end sequencing. scBS-seq: The zona pellucida of MII oocytes and 2-cell embryos were firstly removed by Tyrode's Solution. Construction of the scBS-seq or scCOOL-seq libraries were performed as previously described (Guo et al., 2017a; Smallwood et al., 2014). DNA libraries were sequenced on a Novaseq sequencer for 150 bp paired-end sequencing. Single-cell RNA-seq: Single-cell RNA-seq of Nlrp14-/- MII oocytes and Nlrp14mat△/+ 2-cell embryos were conducted by using smart-seq2 protocol (Picelli et al., 2014), while WT controls were also included. In brief, single cell was picked into lysis buffer and Poly(A)+ RNA was captured by Oligo(dT) primer. The first strand of cDNA was synthesized by reverse transcription, then amplified, purified and fragmented. Fragmented cDNA was processed to construct RNA-seq library by NEBNext UltraII DNA Library Prep Kit for Illumina. ACE-seq, scBS-seq and scRNA-seq
GEO Accession
GSM5662926