GSM8223971: RNA-seq_Oct4_KO_Morula_2; Mus musculus; RNA-Seq
All the embryos for RNA-seq were collected from female mice superovulated with PMSG and hCG. Early and late morula samples were collected at E2.75 and E3.25, respectively. Early and late ICM samples were collected at E2.5 and cultured in KSOM with MEKi (PD0325901, 1 μM) until early and late blastocyst stages, respectively. ICM were collected by immunosurgery. Single-embryo RNA-seq was performed as previously described with some modifications (Kurimoto et al. 2007, Nakamura et al. 2015). 2,493 copies of ERCC RNA Spike-In (Ambion) were added to each single-embryo sample. To remove PCR duplicates, we used R2SP-UMI-d(T)24 primer, instead of V1d(T)24 primer, for reverse transcription (RT). To reduce byproducts derived from the RT primer, the poly(A) tailing reaction was performed only for 1 min at 37°C. cDNA was amplified with V3d(T)24 and R2SP primers and Terra PCR Direct Polymerase (Clontech) for 16 cycles. The cDNA was further amplified for four cycles with NH2-V3d(T)24 and NH2-R2SP primers, and purified thrice with 0.6x AMPure XP beads. The 3' end enriched libraries were constructed using KAPA HyperPlus Library Preparation Kit (VWR International, KK8513) with 6 cycles of PCR. All the oligos used in the RT and library amplification are in Table EV3. The sequencing was performed on the NextSeq 500 system with single-end 75bp.