Supplementary Table S1. Primer sequence information Gene Primer sequence 1 st round PCR 2 nd round PCR Accession# Dnmt3a TCAAGGAGATCATTGATG TGTTCCCACACATGAGCA AGCGGCTGGTGTATGAGG AGCAGATGGTGCAATAGGAC NM_007872.4 Dnmt3b CCTACCCGGAATGAACAG ACAGGCAAAGTAGTCCTTC AGCTCGAGCTGCAGGACT AGATCCTTTCGAGCTCAGTG NM_001003960 Dnmt1 CAGTGGCATGAACCGCTTCA GTCTGGGCCACGCCATACT GCTACTGTGACTACTACCG AAAGGTGCACTGGTAGCC NM_010066.4 Suv39h1 CCATCTACGAGTGCAACTC CTCATCAAGGTTGTCTATG CGCTACGATCTCTGCATCTT ACAAAATGAGAGATGTTGCC NM_011514.2 G9a GACAGAACGCGGGTTTGAG CAGGCCTTATGGAAGCGG AGGGCACAAGTGCATGGC CCCGCTGTGCAGAAGTAG NM_145830.1 Setdb1 CACACGCCAGTTCTATGA TCAGGAAGACCCAGTTCCT ACCTCAATCACAGTTGCAGC CTCCTTGCCTTCCACACTG NM_001163641 Glp TGGTCAAGTATGAGCTGATGC TGCTATGGTCACCTCTTTG ATGGTGAAGCACCAGTGCTG CATTGTTGACTCGAGAGGCA NM_001012518 Bmi1 GAGGGTACTTCATTGATG ACTTTCCGATCCAATCTG GAGACCAGCAAGTATTGTCC CAAGCTTATTATCTCATCATCAG NM_007552.4 Eed CTGAGTGCTGATTATGATC TATGAGGATCTTCTACTTC TCTACCAGAGACATACATAG CCAAATGTCACACTGGCTGT NM_021876.3 Suz12 CTCTATTTCCACAGTGATAC TATCTATTGACATTATGC AAGGAGAGAAAGAAGTGATG TCATGCATGCTGACTAGATG NM_199196.2 Yy1 AAGAAGTGGGAGCAGAAGC TGTTCTTGGAGCATCATC TGCAGATCAAGACCCTGGA GAGTTCTCTCCAATGATCTG NM_009537.3 Jmjd3 CTCACCATCAGCCACTGTG AGCTCTCACAGGGCCAGAT TGCCAGCAAGAATGCCAAG CCAGAGTCTTGGTGGAGAAA NM_001017426 Jhdm2a AGCATTCTTGGCTTTGACA CATCAGATCATCAAACCTG GGCAGCCAGTGATGGTGT CCTTAAGTTTCAACACCATTG NM_001038695
Jhdm3a TTCTACCAGTGTGAGGTG TCTCATTGAAGCGCATGTC CATAGTGAGTCAGGACTGTC AGTCTAGACTTGACTCTCTT NM_001161823 Lsd1 TCTGGCTTGGCAGCAGCTC GTGGCTTCTAGCAACCGGT ACTTCTGGAAGCCAGGGATC CTTGATCTTGGCCAGTTCCA NM_133872.2
Supplementary Table S2. General functions of the reprogramming-related genes analyzed in this study. Gene Relevant information Gene ontology (GO) Dnmt1 Dnmt3a and Dnmt3b Suv39h1 G9a Setdb1 DNA (cytosine-5-)-methyltransferase 1. Dnmt1 has a role in the establishment and regulation of tissue-specific patterns of methylated cytosine residues [1]. Aberrant methylation patterns are associated with certain human tumors and developmental abnormalities [2]. DNA (cytosine-5-)-methyltransferase 3a and 3b. Both are required for genome-wide de novo methylation and is essential for the establishment of DNA methylation patterns during development [3]. Dnmt3a and Dnmt3b play a role in paternal and maternal imprinting [4]. Dnmt3a can actively repress transcription through the recruitment of HDAC activity [5]. Mutations in Dnmt3b cause the immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome [6]. Eight alternatively spliced transcript variants have been described [7]. Suppressor of variegation 3-9 homolog 1. Suv39h1 specifically methylates H3K9, which represents a specific tag for transcriptional repression by recruiting HP1 [8]. It mainly functions in heterochromatin formation at pericentric and telomere regions [9]. Euchromatic histone-lysine N-methyltransferase 2. G9a is a histone methyltransferase that specifically mono- and dimethylates H3K9 in euchromatin [10]. G9a functions in self-renewal of embryonic stem cells [11]. It is also known to repress reprogramming by epigenetically inactivating Oct4 promoter [12] SET domain, bifurcated 1. It regulates histone methylation, gene silencing, and transcriptional repression [13]. Setdb1 possibly participates in maintaining histone methylation pattern through DNA replication [14]. Setdb1 associates with various transcription repressors and with PML nuclear body [15]. (Maintenance) DNA methylation Regulation of gene expression DNA methyltransferase activity DNA/protein/chromatin binding Transcription corepressor activity Histone methyltransferase activity Chromatin modification Heterochromatin formation Histone methyltransferase activity Regulation of gene expression Protein/chromatin binding Histone methyltransferase activity Regulation of gene expression ICM cell proliferation
Glp Bmi1 Eed Yy1 Jmjd3 Jhdm2a Jhmd3a G9a like protein (Euchromatic histone-lysine N-methyltransferase 1). Glp is a histone methyltransferase that specifically mono- and dimethylates H3K9 in euchromatin [16]. This protein may be involved in the silencing of MYC- and E2Fresponsive genes and therefore could play a role in the G0/G1 cell cycle transition [17] B lymphoma Mo-MLV insertion region 1 homolog. Polycomb ring finger oncogene. Bmi1 is a component of polycomb repressive complex 1 (PRC1)-like complex that is required to maintain the transcriptionally repressive state of Hox genes throughout development [18]. Bmi1 mediates H2A119 ubiquitination [19]. Embryonic ectoderm development. Eed encodes a member of the PcG family and interacts with Ezh2 to form polycomb repressive complex 2 (PRC2) [20]. This Ezh2-Eed in PRC2 methylates H3K27, leading to transcriptional repression of target gene [21]. It is involved in X chromosome inactivation [22]. Yin and Yang 1. Yy1 is a master regulator of development [23]. The protein is involved in repressing and activating a diverse number of promoters [24]. Proposed to recruit the PRC2/EED-EZH2 complex to target genes that are transcriptional repressed. Yy1 plays a role in genome integrity [25]. Jumonji domain containing 3. KDM6B. Jmjd3 specifically demethylates di-and trimethylated H3K27 [26]. It plays a central role in regulation of posterior development, by regulating HOX gene expression [27]. Jmjd3 is used by HPV E7 to reprogram host cells [28]. JmjC domain-containing histone demethylation protein 2A. KDM3A. Jmjd1A. Jhdm3a specifically demethylates mono- and dimethylated H3K9 [29]. It is involved in hormone-dependent transcriptional activation [29] and male infertility and obesity [30]. Jumonji C domain-containing histone demethylase 3A. KDM4A. JMJD2A. Jhdm3a specifically demethylates trimethylated H3K9 and H3K36 [31]. Jhdm3a is used in guide-directed target chromatin modification [32] Histone methyltransferase activity Embryo development Chromatin modification Sequence-specific DNA binding Ubiquitin-protein ligase activity Chromatin modification Chromatin modification Regulation of gene expression Protein binding Sequence-specific DNA binding Transcription factor activity Four-way junction DNA binding Histone demethylation Inflammatory response Chromatin modification Histone demethylase activity Hormone-mediated signaling Cell differentiation Histone demethylase activity Chromatin modification
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Figure S1 RT PCR results for 15 reprogramming related genes. cdna from mouse embryonic stem cells (mescs) was used as the template. The size of individual PCR amplicons is indicated.
A B Ct values Figure S2 Results of real time PCRs showing cycle threshold (Ct) values of 15 different transcripts at different PCR cycles. A, PCR strategy. We first pooled five mouse blastocysts and obtained cdnas in a single tube before dividing again into five equal parts. The parts were then used as templates in PCR 1, each with different cycle 25, 30, 35, 40, 45 cycles. Then the PCR 1 products were diluted and used in real time single gene PCR to obtain Ct values of the 15 gene transcripts. For each transcript, real time PCR was performed three times (x3). Basically, we have used the same one step RT PCR reagents, PCR 1 and PCR 2 primers, and nested PCR strategy in Fig. 1. B, Ct values of each transcript at different PCR cycles. Ct values are indicated above the bars. Red lines denote predicted trend lines with anticipation. Error bars, standard deviation.
Figure S3 Raw gene expression data that are not corrected for the Lsd1 expression (A) and coefficient of variation (CV) of gene expression (B) are shown. Error bars denote the standard deviation. The average expression levels and standard deviations of individual blastocyst groups in mouse are included for the CV calculation.
Figure S4 Comparison of GEPs from donor cumulus cells and SCNT blastocysts carrying the donor genome. A, Gene expression levels. B, Spline plots of GEPs. Upper, donor cell profile; lower, SCNT profile. Note that Dnmt3b is set as the reference because the Lsd1 expression is relatively low in mouse cumulus cells compared to embryonic Lsd1 expression and thus is unsuitable for a standard.
Figure S5 Single gene PCR of the reprogramming related genes. The gene expression level was examined by single gene PCR using pooled first round PCR products as templates.
Figure S6 Single gene RT PCR for Dnmt1, Setdb1, and Jhdm3a. Single gene RT PCR was performed using cdnas from single blastocysts that were freshly prepared from in vivo (n = 12) and SCNT blastocysts (n = 12) as templates.