KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal anti-5hmC | Active Motif | Cat # 39769; RRID:AB_10013602 |
| Rabbit polyclonal anti-5caC | Active Motif | Cat # 61225; RRID:AB_2793557 |
| Goat anti-rabbit IgG-HRP | Santa Cruz Biotechnology | Cat # sc-2004; RRID:AB_631746 |
| Mouse monoclonal anti-FLAG M2 | Sigma-Aldrich | Cat # F1804; RRID:AB_262044 |
| Rabbit monoclonal anti-GAPDH | Cell Signaling Technology | Cat # 2118S; RRID:AB_561053 |
| Mouse IgGκ BP-HRP | Santa Cruz Biotechnology | Cat # sc-516102; RRID:AB_2687626 |
| Goat anti-rabbit IgG-HRP | Cell Signaling Technology | Cat # 7074S; RRID:AB_2099233 |
| Rabbit polyclonal anti-Nanog | Abcam | Cat # ab80892; RRID:AB_2150114 |
| Goat anti-rabbit IgG H&L Alexa Fluor 488 | Abcam | Cat # 150081; AB_2734747 |
| SSEA1 eFluor 660 monoclonal antibody | Invitrogen | Cat # 50–8813-41; RRID:AB_11217669 |
| Biological Samples | ||
| Unmethylated lambda phage DNA | Promega | Cat # D152A |
| Methylated lambda phage DNA | Schutsky et al., 2018 | N/A |
| T4 phage DNA | Schutsky et al., 2018 | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| T4 Polynucleotide Kinase | NEB | Cat # M0201S |
| GlutaMAX | Gibco | Cat # 35050061 |
| Fetal bovine serum (FBS) | TCB | Cat # 101 |
| Penicilin / Streptomycin | Gibco | Cat # 15140122 |
| Heat-inactivated FBS | Gibco | Cat # 10082147 |
| MEM non-essential amino acids | Gibco | Cat # 11140050 |
| Sodium pyruvate (cell culture) | Gibco | Cat # 11360070 |
| 2-Mercaptoethanol (cell culture) | Gibco | Cat # 21985023 |
| ESGRO recombinant mouse LIF | Sigma-Aldrich | Cat # ESG1107 |
| PD0325901 | Stemolecule | Cat # 04–0006 |
| CHIR99021 | Sigma-Aldrich | Cat # SML1046 |
| Doxycycline (cell culture) | Stem Cell | Cat # 72742 |
| Lipofectamine 3000 Transfection Reagent | Invitrogen | Cat # L3000001 |
| Cytobuster Protein Extraction Reagent | Millipore | Cat # 71009 |
| Immobilon HRP Chemiluminescent Substrate | Millipore | Cat # WBKLS0100 |
| Nucleoside Digestion Mix | NEB | Cat # M0649S |
| Geneticin selective antibiotic | Gibco | Cat # 10131035 |
| Puromycin selective antibiotic | Takara Bio | Cat # 631305 |
| Polybrene | American Bio | Cat # AB01643–00001 |
| Triton X-100 | Supelco | Cat # TX1568–1 |
| Bovine serum albumin, fraction V | Sigma-Aldrich | Cat # 12659 |
| DNase I recombinant | Roche | Cat # 4716728001 |
| Superscript III Reverse Transcriptase | Invitrogen | Cat # 18080–044 |
| RPMI media | Gibco | Cat # 11875119 |
| TDE1 transposase | Illumina | Cat # 15027865 |
| 2’-Deoxycytidine-5-Carboxylic acid sodium salt (LC/MS-MS) | Berry & Associates | Cat # PY 7593 |
| 5-Formyl-2’-deoxycytidine (LC/MS-MS) | Berry & Associates | Cat # PY 7589 |
| 5-Hydroxymethyl-2’-deoxycytidine (LC/MS-MS) | Berry & Associates | Cat # PY 7588 |
| 5-Methyl-2′-deoxycytidine Hydrochloride (LC/MS-MS) | Berry & Associates | Cat # PY 7635 |
| Critical Commercial Assays | ||
| Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit | Invitrogen | Cat # C10424 |
| Dead Cell Apoptosis Kit with Annexin V FITC and PI | Invitrogen | Cat # V13242 |
| StemAb Alkaline Phosphatase Staining Kit II | Stemgent | Cat # 00–0055 |
| Power SYBR Green Master Mix (qRT-PCR) | Applied Biosystems | Cat # 4367659 |
| Recombinant MBP-APOBEC3A (A3A)-His | Schutsky et al., 2018; This paper | N/A |
| CpG Methyltransferase (M.SssI) | NEB | Cat # M0226S |
| NEBNext Library Quant Kit | NEB | Cat # E7630 |
| Bioanalyzer High-Sensitivity DNA chip | Agilent | Cat # 5067–4626) |
| High Output Kit v2.5 (75 cycles) | Illumina | Cat # 20024906 |
| Bioanalyzer DNA 1000 chip | Agilent | Cat # 5067–1504 |
| MiSeq Reagent Nano Kit v2 (500 cycles) | Illumina | Cat # MS-103–1003 |
| Deposited Data | ||
| 5hMeDIP-seq (iPSC day 4) | Sardina et al., 2018 | GSM3315344; GSM3315345 |
| RNA-seq (iPSC day 5) | This paper | GSE158334 |
| ATAC-seq (iPSC day 5 and 10) | This paper | GSE158334 |
| Caldwell et al., 2021 Original Data | This paper | DOI: 10.17632/42kc23sjy9.1 |
| Experimental Models: Cell Lines | ||
| Human: HEK293T cells | ATCC | Cat # CRL-11268; RRID:CVCL_1926 |
| Mouse: NIH/3T3 cells | ATCC | Cat # CRL-1658; RRID:CVCL_0594 |
| Human: PLAT-E retroviral packaging cells | Cell Bio Labs | Cat # RV-101; RRID:CVCL_B488 |
| Mouse: CF1 Mito-C feeder cells | This paper | N/A |
| Mouse: KH2-OKSM; rosa26:M2rtTA (OKSM; rtTA) mESCs | Stadtfeld et al., 2010 | N/A |
| Mouse: WT and Tet2−/− OKSM; rtTA primary MEFs | This paper | N/A |
| Oligonucleotides | ||
| Mouse Tet1 / Tet2 cloning primers (See Table S2 for primer sequences) | This paper | N/A |
| iPSC reprogramming qRT-PCR primers (See Table S2 for primer sequences) | This paper; Hu et al., 2014 | N/A |
| bACE-pyroseq/MiSeq primers (See Table S3 for primer sequences and genomic coordinates) | This paper | N/A |
| bACE 5mC/5hmC spike-in oligonucleotide (See Figure S3A for oligo info) | This paper | N/A |
| Recombinant DNA | ||
| Tet1-CD WT and T1642 / Y2049 / HxD mutant pLEXm expression vectors (See Table S1 for full list) | This paper | N/A |
| Tet2-CD WT and T1285 / HxD pLEXm expression vectors (See Table S1 for full list) | This paper | N/A |
| Tet1-CD WT and T1642V / HxD pMXs retrovirus vectors | This paper | N/A |
| Tet2-CD WT and HxD pMXs retrovirus vectors | Hu et al., 2014 | N/A |
| Tet2-CD T1285A/E mutant pMXs retrovirus vectors | This paper | N/A |
| Tet2 Int8a pX330 CRISPR vector | This paper | N/A |
| Tet2 Int10a pX330 CRISPR vector | This paper | N/A |
| ploxPneo-1 | Nagy et al., 1998 | N/A |
| Software and Algorithms | ||
| Fiji | Schinderlin et al., 2012 | https://imagej.net/Fiji/Downloads |
| STAR (version 2.6.1a) | Dobin et al., 2013 | https://github.com/alexdobin/STAR |
| Bowtie2 (version 2.2.5) | Langmead & Salzberg, 2012 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml |
| SAMTools (version 1.7) | Li et al., 2009 | http://samtools.sourceforge.net/ |
| BEDTools (version 2.27.1) | Quinlan & Hall, 2010 | https://github.com/arq5x/bedtools2/releases |
| featureCounts (version 1.6.2) | Liao et al., 2014 | https://www.rdocumentation.org/packages/Rsubread/versions/1.22.2/topics/featureCounts |
| DESeq2 (version 1.26.0) | Love et al., 2014 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html |
| clusterProfiler (version 3.14.3) | Yu et al., 2012 | https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html |
| ChIPseekers (version 1.22.1) | Yu et al., 2015 | https://www.bioconductor.org/packages/release/bioc/html/ChIPseeker.html |
| Bigwig tracks | Kent et al., 2010 | http://genome.ucsc.edu |
| macs2 (version 2.2.7.1) | Zhang et al., 2008 | https://github.com/taoliu/MACS/wiki/Install-macs2 |
| Bedops (version 2.4.39) | Neph et al., 2012 | https://bedops.readthedocs.io/en/latest/content/installation.html |
| GenomicRanges (R package) | Lawrence et al., 2013 | https://bioconductor.org/packages/release/bioc/html/GenomicRanges.html |
| Pheatmap (R package) | Raivo Kolde | https://www.rdocumentation.org/packages/pheatmap |
| Trim Galore (version 0.6.4) | Felix Krueger | http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) |
| Bismark (version 0.22.3) | Felix Krueger | https://www.bioinformatics.babraham.ac.uk/projects/bismark |
| GraphPad Prism 8 (version 8.4.1) | GraphPad Software | www.graphpad.com |
| Other | ||
| Agilent QuikChange Lightning Site-Directed Mutagenesis Kit | Agilent | Cat # 210519 |
| RNeasy Mini Kit | Qiagen | Cat # 74104 |
| KAPA Stranded mRNA-Seq Kit (KK8420) | Roche | Cat # 07962193001 |
| KAPA Single-Indexed Adaptor Kit (KK8701) | Roche | Cat # 0800570200 |
| KAPA Library Quantification Kit (KK4924) | Roche | Cat # 07960140001 |
| EpiTect Bisulfite Kit | Qiagen | Cat # 59104 |
| Zymo Oligo Clean & Concentrator Kit | Zymo Research | Cat # D4060 |
| Pyromark PCR Kit | Qiagen | Cat # 978703 |
| MinElute Reaction Clean-up Kit | Qiagen | Cat # 28204 |
| AMPure XP | Beckman Coulter | Cat # A63881 |
| Multiplex PCR Kit | Qiagen | Cat # 206143 |