Supporting Information Li et al. 10.1073/pnas.1617802113 SI Materials and Methods transfection. Cells were transfected with reporter constructs TOP- Plasmids. Mutant Tet3 (H950D and Y952A) was generated by or FOP-Flash using Lipofectamine 2000 (Invitrogen). Cell extracts PCR using Pfu polymerase, Dpn I treatment, and transformation were prepared 48 h after transfection. The luciferase activity was (Stratagene). The ORF of mouse Tet3, mutant Tet3 (H950D and evaluated by using the Dual-Luciferase Reporter Assay System ’ Y952A), Sfpr4, Pcdha4, and Pcdha7 were cloned into pPyCAGIP (Promega) according to the manufacturer s recommendations. vector (60). qRT-PCR. qRT-PCR was performed by using Universal SYBR Green Generation of Tet3 KO and Tet1/2/3 TKO mESCs. Tet3 KO and Tet1/2/3 Master Mix (Roche) and analyzed by a StepOne Plus real-time PCR ’ TKO mouse ESCs were generated from mice (C57BL/6 back- system (Applied Biosystems), according to the manufacturer sin- ground) bearing the individual floxed alleles (Fig. S1C) (14, 18), structions, and the data were normalized for Gapdh expression. followed by excision of the floxed exon by transient expression of The primers used for qRT-PCR are listed in Dataset S4. Cre recombinase. Immunohistochemistry. Immunohistochemistry was performed as mESC Culture and Differentiation. mESCs were maintained on mi- described (61) with the primary antibodies described below. For ∼ tomycin C-treated mouse embryonic fibroblasts (MEFs; feeders) in statistical analysis, 300 cells were examined for each experiment, standard medium (61). Neural differentiation in SFEB culture was which was repeated four times. Mouse embryos were fixed over- night in 4% (wt/vol) paraformaldehyde, saturated with 20% (wt/vol) performed as described (31) with a minor modification. Briefly, μ mESCs were dissociated to single cells in 0.25% trypsin–EDTA sucrose, and frozen in O.C.T. embedding medium, and 8- msec- (Invitrogen). Dissociated ESCs (1 × 105 cells per mL) were seeded tions were prepared on a cryotome. The following antibodies were onto a nonadhesive bacterial-grade dish and cultured in Knockout used: anti-Sox1 (no. 4194; Cell Signaling), anti-Sox2 (NL20181V; DMEM supplemented with 10% (vol/vol) KSR, 2 mM glutamine, R&D), anti-cTnT (CT3; Hybridoma Bank), anti-T (Brachyury) (AF2085; R&D), anti-Gata4 (sc-1237; Santa Cruz), anti-Tbx6 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol (AF4744; R&D), anti-Isl1 (NL1837R; R&D), anti-Foxa2 (2-ME). On day 2, the medium was changed to N2B27 medium (IC2400G; R&D), and anti–Active-β-Catenin (05-665; Millipore) (62). The medium was changed every other day. The day on which generated against the peptide 36-44, HSGATTTAP, dephosphory- ESCs were seeded to differentiate is defined as differentiation day lated at Ser-37 and Thr-41. 0. Because Tet1/2/3 TKO mESCs did not differentiate well under SFEB culture in N2B27 medium, they were differentiated by using Whole-Transcriptome RNA-Seq. Total RNA was extracted by using an the SFEB method in 10% (vol/vol) KSR medium continuously. RNeasy Plus Mini Kit (Qiagen). Multiplexed libraries were con- For nonpermissive neural differentiation, dissociated ESCs (1 × 5 structed by using a SOLiD Total RNA-seq kit. Libraries were 10 cells per mL) were seeded onto an ultralow attachment culture sequenced on the SOLiD 5500 platforms according to the man- dish (Corning) and cultured in Knockout DMEM supplemented ufacturer’s instructions. Approximately 10–15 million uniquely with 15% (vol/vol) FBS, 2 mM glutamine, 0.1 mM nonessential mapped reads per sample were generated. Reads were aligned amino acids, and 0.1 mM 2-ME for 4 or 7 d. to the mouse genome (mm9; NCBI37) by using Tophat. DE-Seq was performed to identify differentially expressed genes. GO Tet1 Generation of Tet1/2/3-Deficient Mice. The gene was inactivated biological process analysis was performed by using DAVID Bio- – by targeting its exons 8 10 encoding part of the catalytic domain informatics Resources 6.7 (https://david.ncifcrf.gov/). Pathway anal- Tet2 The Tet3 (18). targeting was described (14). gene was in- yses were performed by using Ingenuity Pathway Analysis software. activated by targeting exon 2 (18). The deleted allele bears a frame-shift mutation that results in a truncated protein (Fig. S1B). ChIP-Seq. ChIP using anti-Tet3 antibody (gift of Guoliang Xu, The strains of Zp3-Cre and Stra8-Cre mice used in this study are Institute of Biochemistry and Cell Biology, Shanghai, China) FVB/N-TgN(Zp3-Cre)3Mrt and Tg(Stra8-Cre)1Reb/J (Jackson (16) was performed in NPCs. To generate NPCs, mESCs were Laboratories) respectively. Zp3-Cre and Stra8-Cre are exclusively differentiated in SFEB culture conditions [10% (vol/vol) KSR] expressed in growing oocytes and spermatogonia, respectively (63, for 5 d, followed by adherent culture for 6 d in N2B27 with 10 ng/mL 64). We generated Tet1/2/3 triple-deficient mice by crossing Zp3- EGF and basic FGF. ChIP assays were carried out as described fl/fl Cre and Stra8-Cre mice with Tet1/2/3 mice to generate mice in (5). Briefly, chromatin was sheared by using truChIP High which expression of all three Tet proteins was abrogated in oocytes Cell Chromatin Shearing Kit with Nonionic Shearing Buffer fl/fl and sperm, respectively. The progeny of Tet1/2/3 Zp3-Cre female (Covaris). Chromatin fragments from two biologically independent fl/fl and Tet1/2/3 Stra8-Cre male lack all three Tet proteins beginning at NPCs were immunoprecipitated by using anti-Tet3 antibody. the zygotic stage. Tet1/2/3 triple-deficient mice were on C57BL/6 ChIP-seq libraries were constructed by using a 5500 SOLiD background. Fragment 48 Library Core Kit. Libraries were sequenced on the SOLiD 5500 platforms, according to the manufacturer’s in- Whole-Mount in Situ Hybridization. Embryos from E9.25 were dis- structions. Sequencing reads were mapped against mm9 by using sected, fixed at 4 °C overnight in 4% (wt/vol) paraformaldehyde/ Bowtie. Tet3-enriched regions were identified by MACS peak- PBS, dehydrated through a series of increasing methanol con- calling software (Version 1.4.2) (66). We had performed pulldowns centrations, and stored at −20 °C. Whole-mount in situ hybrid- by using control IgG antibody, but no chromatin was recovered. ization was performed as described (65). Tet3 probe was amplified Therefore, sequencing reads from input were used as negative from mouse cDNA (GenBank accession no. NM_183138.2) and controls in MACS. The statistical cutoff used for identifying corresponds to the nucleotide 569–1,443 region. Tet3-binding sites was P < 0.0005. Genomic distribution of Tet3- bound sites was performed by using the cis-regulatory element Luciferase Reporter Assay. β-catenin/TCF reporter assay was per- annotation system (67). The distribution of Tet3-binding sites to formed as described (61). Briefly, cells were seeded at a density of TSS was undertaken by using ChIPseek (68). De novo motif 1 × 105 per well into a 24-well tissue-culture plate 24 h before discovery was performed by using the findMotifsGenome.pl Li et al. www.pnas.org/cgi/content/short/1617802113 1of9 function of the Homer software package (69) using default pa- using BS-Seeker2 (71). The primer sequences are shown in rameter settings. Tet3-specific peaks within 100 kb flanking the Dataset S4. TSS were used for motif analysis. Single-Embryo RNA Sequencing. CTL and Tet1/2/3 TKO embryos BS-Seq. BS-seq was performed as described (70). Briefly, genomic were collected at E6.5. Single embryos were lysed and converted DNA was extracted by using PureLink Genomic DNA mini Kit into double-stranded cDNA by using the SMARTer ultralow (Invitrogen) and treated with sodium bisulfite (MethylCode Bi- RNA kit for Illumina sequencing (Clontech). A total of 1 ng of sulfite Conversion Kit; Invitrogen). The PCR amplicons were cDNA for each sample was used for preparing libraries by using generated by using a PyroMark PCR kit (Qiagen) and quantified by the Nextera XT DNA sample preparation kit (IIlumina). The using a Quant-iT PicoGreen dsDNA reagent (Invitrogen). The good quality of the prepared libraries was validated by using the PCR amplicon was generated by using a PyroMark PCR kit Bioanalyzer high-sensitivity DNA kit (Agilent). Libraries were μ sequenced on Illumina HiSeq 2500 platforms according to the (Qiagen). PCR amplicons were then mixed together for 1 g final ’ – quantity and used for the library preparation using NEBNext manufacturer s instructions. Approximately 10 15 million DNA Library Modules for illumine platform (NEB). The final uniquely mapped reads per sample were generated. Reads were aligned to the mouse genome (mm9; NCBI37) by using Tophat. libraries were then combined together and quantified by using a DE-Seq was performed to identify differentially expressed genes. KAPA library quantification kit for Illumina (KAPA Biosystems), GO biological process analysis was performed by using DAVID then sequenced on Miseq (300 bp, paired end; Illumina). The data Bioinformatics Resources 6.7 (https://david.ncifcrf.gov/). are based on thousands of sequence reads per amplicon. To monitor bisulfite conversion efficiency, a 210-bp spike-in control Statistical Analysis. All values are shown as means ± SD. To de- was generated by using unmethylated lambda DNA (Promega) as termine the significance between groups, comparison was made by a template and added to the genomic DNA to a final ratio of using Student’s t test. For all statistical tests, the 0.05 confidence 0.5%. Based on C-to-T conversion efficiencies of spike-in controls, level was considered statistically significant. In all figures, * de- the average bisulfite conversion efficiencies were >99.5%. Reads notes P < 0.05 and ** denotes P < 0.01 in a two-tailed Student’s were aligned to the genome and used to measure methylation t test.