Extended Data Fig. 5: Further analyses of ruminant haSCs about formative features.
From: Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells
a. The expression of typical naïve-, formative-, and primed-pluripotency genes in b-haSCs at the single-cell level. b. Heatmap displaying the expression of naïve-, formative-, and primed-pluripotency genes in b-haSCs sequenced by scRNA-seq. c. Hierarchical clustering analysis of b-haSCs, o-haSCs, primed b-SCs, and primed o-SCs based on the global transcriptome. d. RT-PCR analysis of core formative markers (DNMT3A, DNMT3B, OTX2) and pluripotency marker (OCT4) in b-haSCs, o-haSCs, and formative m-SCs. M, DNA marker. n = 3 independent experiments with similar results. e. Western blots analysis of core formative markers (DNMT3A, DNMT3B, OTX2) and pluripotency marker (OCT4) in b-haSCs, o-haSCs, and formative m-SCs. n = 3 independent experiments with similar results. f. Scatter-plots showing the reproducibility of CUT&Tag assay between different biological replicates of b-haSCs. The Pearson’s correlation coefficients (Cor.) are shown. g. UCSC genome browser views showing H3K4me3 and H3K27me3 tracks of representative formative- and primed-pluripotency genes in b-haSCs and primed b-SCs. h. Hierarchical clustering analysis of b-haSCs and primed b-SCs based on the H3K4me3 and H3K27me3 CUT&Tag signals. i. UCSC genome browser views showing H3K4me3 and H3K27me3 tracks of selected pluripotency-associated genes in b-haSCs and primed b-SCs. Note that H3K4me3 but not H3K27me3 signals exhibited high enrichment levels in both b-haSCs and primed b-SCs. j, k. Bisulfite pyrosequencing analysis of paternally (a) and maternally (b) imprinted regions in b-haSCs and o-haSCs. The filled and open squares represent methylated and unmethylated CpG sites, respectively. l. Whole genome bisulfite sequencing (WGBS) analysis of known imprinting control regions in b-haSCs. RNA-seq data of o-haSCs and b-haSCs generated in this study, previously published data for bovine primed-b-SCs are from ref. 13, PMID: 29440377 (GSE110040), diploid primed o-SCs are from ref. 44, PMID: 33065542 (PRJNA609175). The H3K4me3 and H3K27me3 data of b-haSCs generated in this study, previously published data for diploid primed b-SCs are from ref. 13, PMID: 29440377 (GSE110040).
