|
Antibodies
|
| Goat polyclonal anti-GFP |
Rockland |
Cat#600101215; RRID: AB 11181883 |
| Rabbit polyclonal anti-Cleaved caspase3 |
Cell Signaling Technology |
Cat#9661; RRID: AB_2341188 |
| Rat polyclonal anti-Ctip2 |
Abcam |
Cat#ab18465; RRID: AB_2064130 |
| Rabbit polyclonal anti-Cux1 |
Santa Cruz |
Cat#sc-13024; RRID: AB_2261231 |
| Guinea pig polyclonal anti-DCX |
EMD Millipore |
Cat#AB2253; RRID: AB_1586992 |
| Rabbit monoclonal anti-GAPDH |
Cell Signaling Technology |
Cat#2118; RRID: AB_561053 |
| Rat polyclonal anti-HA |
Roche |
Cat#11867423001; RRID: AB_390918 |
| Rabbit polyclonal anti-Histone H3 |
Abcam |
Cat#ab1791; RRID: AB_302613 |
| Mouse monoclonal anti-Ki67 |
BD Biosciences |
Cat#550609; RRID: AB_393778 |
| Rabbit polyclonal anti-Mettl8 |
Abcam |
Cat#ab122273; RRID: AB_11127706 |
| Rabbit polyclonal anti-mt-ATP6 |
Sigma-Aldrich |
Cat#SAB5700851; |
| Rabbit polyclonal anti-mt-ATP6 |
Proteintech |
Cat#55313-1-AP; RRID: AB_2881305 |
| Mouse monoclonal anti-mt-Co1 (Immunostaining) |
Abcam |
Cat#ab14705; RRID: AB_2084810 |
| Mouse monoclonal anti-mt-Co1 (WB) |
Thermo Fisher Scientific |
Cat#459600; RRID: AB_1501840 |
| Rabbit polyclonal anti-mt-Co2 |
Proteintech |
Cat#55070-1-AP; RRID: AB_10859832 |
| Chicken polyclonal anti-Nestin |
Aves labs |
Cat#NES; RRID: AB_2314882 |
| Mouse monoclonal anti-Pax6 |
BD Biosciences |
Cat#561462; RRID: AB_10715442 |
| Rabbit polyclonal anti-Pax6 |
BioLegend |
Cat#901301; RRID: AB_2565003 |
| Mouse monoclonal anti-Satb2 |
Abcam |
Cat#ab51502; RRID: AB_882455 |
| Goat polyclonal anti-Sox2 |
R&D system |
Cat#AF2018; RRID: AB_355110 |
| Rabbit monoclonal anti-TBR2 |
Abcam |
Cat#ab183991; RRID: AB_2721040 |
| Rabbit polyclonal anti-Tomm20 |
THOMAS SCIENTIFIC LLC |
Cat#11802-1-AP; RRID: AB_2207530 |
| Mouse monoclonal anti-α-tubulin |
Cell Signaling Technology |
Cat#3873S; RRID: AB_1904178 |
| Mouse monoclonal anti-β-actin |
Thermo Fisher Scientific |
Cat#AM4302; RRID: AB_2536382 |
| Donkey polyclonal anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Thermo Fisher Scientific |
Cat#A-11055; RRID: AB_2534102 |
| Donkey polyclonal anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 |
Thermo Fisher Scientific |
Cat#A-21447; RRID: AB_2535864 |
| Donkey polyclonal anti-Guinea Pig IgG (H+L) AffiniPure Secondary Antibody, Alexa Fluor 488 |
Jackson ImmunoResearch |
Cat#706-545-148; RRID: AB_2340472 |
| Donkey polyclonal anti-Guinea Pig IgG (H+L) AffiniPure Secondary Antibody, Alexa Fluor 647 |
Jackson ImmunoResearch |
Cat#706-605-148; RRID: AB_2340476 |
| Donkey polyclonal anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Thermo Fisher Scientific |
Cat#A-21202; RRID: AB_141607 |
| Donkey polyclonal anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 |
Thermo Fisher Scientific |
Cat#A-10037; RRID: AB_2534013 |
| Donkey polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Thermo Fisher Scientific |
Cat#A-21206; RRID: AB_2535792 |
| Donkey polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 |
Thermo Fisher Scientific |
Cat#A-10042; RRID: AB_2534017 |
| Donkey polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 |
Thermo Fisher Scientific |
Cat#A-31573; RRID: AB_2536183 |
| Donkey polyclonal anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Thermo Fisher Scientific |
Cat#A-21208; RRID: AB_2535794 |
| Donkey polyclonal anti-Chicken IgY (IgG, H+L) AffiniPure Secondary Antibody, Alexa Fluor 488 |
Jackson ImmunoResearch |
Cat#703-545-155; RRID: AB_2340375 |
| Anti-mouse IgG, HRP-linked Antibody |
Cell Signaling Technology |
Cat#7076, RRID: AB_330924 |
| Anti-rabbit IgG, HRP-linked Antibody |
Cell Signaling Technology |
Cat#7074, RRID:AB_2099233 |
|
Bacterial and virus strains
|
| Retro-pSUBGW-U6-Ub-GFP |
(Zhang et al., 2016) |
N/A |
|
Chemicals, peptides, and recombinant proteins
|
| 0.05% (W/V) Trypsin-EDTA |
Corning |
Cat#MT25052CI |
| 10X RT buffer |
Thermo Fisher Scientific |
Cat#53032LT |
| 2-Mercaptoethanol |
Thermo Fisher Scientific |
Cat#21985023 |
| Advantage UltraPure PCR deoxynucleotide mix (10mM each dNTP) |
Takara Bio |
Cat#639125 |
| AMPure XP beads |
Beckman Coulter |
Cat#A63880 |
| AMV Reverse Transcriptase |
Takara Bio |
Cat#2630A |
| Aniline |
THOMAS SCIENTIFIC LLC |
Cat#242284 |
| Antarctic Phosphatase |
New England Biolabs |
Cat#M0289S |
| Aqua-Mount Mounting Medium |
EMSCO/FISHER |
Cat#NC9428056 |
| B-27® Supplement |
Gibco |
Cat#17504044 |
| Blue Protein Loading sample buffer |
New England Biolabs |
Cat#B7703S |
| CHIR-99021 |
StemCell Technologies |
Cat#72054 |
| Chloramphenicol |
Sigma-Aldrich |
Cat#C0378 |
| Chloroform |
Sigma-Aldrich |
Cat#C2432 |
| cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail |
Sigma-Aldrich |
Cat#11836170001 |
| Cy5 conjugated Streptavidin |
Jackson ImmunoResearch |
Cat#016-170-084 |
| DAPI |
BD Biosciences |
Cat#564907 |
| DBCO-PEG4-biotin |
Sigma-Aldrich |
Cat#760749 |
| DirectPCR reagent |
Fisher Scientific |
Cat#NC9724951 |
| DL-Dithiothreitol solution |
Millipore Sigma |
Cat#43816 |
| Dulbecco’s Modification of Eagle’s Medium (DMEM) |
Corning |
Cat#10-013 |
| Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) |
Gibco |
Cat#11330057 |
| Dulbecco’s Phosphate-Buffered Saline (DPBS) |
Corning |
Cat#14190250 |
| EcoRI-HF restriction enzyme |
New England Biolabs Inc |
Cat#R3101S |
| EdU |
Thermo Fisher Scientific |
Cat#E10187
|
| EZ-Tn5 Transposase |
Lucigen |
Cat#TNP92110
|
| Fast Green |
VWR Chemicals |
Cat#E772-25G |
| Fast SYBR Green Master Mix |
Thermo Fisher Scientific |
Cat#4385612 |
| Fetal Bovine Serum (FBS) |
Corning |
Cat#35-010-CV |
| Formaldehyde, 16%, methanol free, Ultra Pure |
Polysciences |
Cat#18814-10 |
| Gibco DMEM, high glucose, no glutamine, no methionine, no cystine |
EMSCO/FISHER |
Cat#21013024 |
| GlutaMAX supplement |
GIBCO |
Cat#35050061 |
| Heparin solution |
StemCell tech |
Cat#7980 |
| Hibernate A Low Fluorescence buffer |
Brain Bits |
Cat#HALF100 |
| Human EGF |
Peprotech |
Cat#AF-100-15 |
| Human insulin solution |
Sigma-Aldrich |
Cat#I9278 |
| Hydrazine |
THOMAS SCIENTIFIC LLC |
Cat#215155 |
| KAPA HiFi Hotstart Readymix |
Emsco/Fisher |
Cat#KK2601 |
| KnockOut Serum Replacement |
Thermo Fisher Scientific |
Cat#10828028 |
| L-Azidohomoalanine (AHA) |
EMSCO/FISHER |
Cat#C10102
|
| LDN-193189 |
StemCell Technologies |
Cat#72147 |
| Lipofectamine 2000 |
Invitrogen |
Cat#11668019 |
| Lipofectamine Stem Reagent |
Fisher Scientific |
Cat#STEM00003 |
| Matrigel (hESC-qualified) |
Corning |
Cat#8774552 |
| MEM Non-Essential Amino Acids Solution |
Gibco |
Cat#11140050 |
| MitoTracker™ Orange CMTMRos |
Thermofisher scientific |
Cat#M7510 |
| mTeSR Plus |
StemCell Technologies |
Cat#5825 |
| N-2 Supplement (100X) |
Thermo Fisher Scientific |
Cat#17502048 |
| Neurobasal medium |
Thermo Fisher Scientific |
Cat#21103049 |
| Not1 restriction enzyme |
New England Biolabs Inc |
Cat#R0189S |
| Nuclease-Free Water (not DEPC-Treated) |
Thermo Fisher Scientific |
Cat#AM9937 |
| Penicillin-Streptomycin |
Thermo Fisher Scientific |
Cat#15140163 |
| PhosSTOP |
Sigma-Aldrich |
Cat#4906845001 |
| Pierce™ ECL Western Blotting Substrate |
Thermo Fisher Scientific |
Cat#PI32109 |
| Piracetam |
Sigma-Aldrich |
Cat#P5295 |
| Polybrene |
Santa Cruz |
Cat#sc-134220 |
| Poly-D-Lysine |
Sigma-Aldrich |
Cat#P6407 |
| Polyethylene glycol (PEG) solution, 40% |
Sigma-Aldrich |
Cat# P1458 |
| Protease Inhibitor Cocktail |
Sigma-Aldrich |
Cat#P8340 |
| Proteinase K |
QIAGEN |
Cat#19133 |
| Puromycin, Dihydrochloride |
EMD MILLIPORE |
Cat#540411 |
| Recombinant Human FGF-basic |
Peprotech |
Cat#100-18B |
| ReLeSR reagent |
StemcellTech |
Cat#5872 |
| RIPA lysis buffer |
Thermofisher |
Cat#89900 |
| RNase Inhibitor, Murine |
New England Biolabs |
Cat#M0314S |
| SB-431542 |
StemCell Technologies |
Cat#72234 |
| SDS (10% w/v) |
Fisher Scientific |
Cat#50-751-7490 |
| Seahorse XF Media & Calibrant |
Agilent |
Cat#103680-100 |
| Seahorse XFe96 FluxPak mini |
Agilent |
Cat#102601 |
| SMARTScribe Reverse Transcriptase |
Takara Bio |
Cat#639537 |
| StemCell freezing media |
ATCC |
Cat#ACS-3020 |
| StemPro Accutase Cell Dissociation Reagent |
Thermo Fisher Scientific |
Cat#A1110501 |
| Streptavidin-HRP Conjugate |
PerkinElmer |
Cat#NEL750001EA |
| Sucrose |
VWR Chemicals |
Cat#97061-430 |
| T4 Polynucleotide Kinase |
New England Biolabs |
Cat#M0289S |
| Target Retrieval Solution |
Agilent Dako |
Cat#S1699 |
| Triton X-100 |
Sigma-Aldrich |
Cat#T8787 |
| TRIzol reagent |
Thermo Fisher Scientific |
Cat#15596026 |
| TrypLE |
Fisher scientific |
Cat#12604013 |
| TWEEN 20 |
Sigma-Aldrich |
Cat#P1379 |
| XF DMEM medium pH 7.4 |
Agilent |
Cat#103575 |
| Y-27632 |
StemCell Technologies |
Cat#72304 |
|
Critical commercial assays
|
| Advantage 2 PCR Kit |
Takara Bio |
Cat#639206 |
| Cell Fractionation Kit |
Abcam |
Cat#ab109719 |
| Click-iT Plus EdU Alexa Fluor 647 Imaging Kit |
Thermo Fisher Scientific |
Cat#C10640
|
| EZ-PCR Mycoplasma test kit |
Biological Industries |
Cat#2070020 |
| KAPA Library Quantification Kit for Illumina NGS |
Kapa Biosystems |
Cat#KK4835 |
| NEBNext Magnesium RNA Fragmentation Modules |
New England Biolabs |
Cat#E6150S |
| NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1) |
New England Biolabs |
Cat#E7300S |
| NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) |
New England Biolabs |
Cat#E7405S |
| NextSeq High Output v2 75 Cycles |
Illumina |
Cat#20024906 |
| Pierce™ Rapid Gold BCA Protein Assay Kit |
Thermo Fisher Scientific |
Cat#A53226
|
| Qubit dsDNA HS Assay Kit |
Thermo Fisher Scientific |
Cat#Q33231
|
| RNA Clean & Concentrator 5 |
Zymo Research |
Cat#R1013 |
| Seahorse XF Cell Mito Stress Test Kit |
Agilent |
Cat#103015 |
| SuperScript III First-Strand Synthesis System |
Thermo Fisher Scientific |
Cat#18080051 |
|
Deposited data
|
| Raw and analyzed RNA-seq and m3C-HAC-seq data |
This paper |
GEO: GSE214445
|
| Code for HAC-seq analysis |
This paper |
https://doi.org/10.5281/zenodo.7535193
|
|
Experimental models: Cell lines
|
| Mouse: Neuro-2a Neuroblastoma (N2a) cell line |
ATCC |
Cat#CCL-131 |
| Human: C1-2 iPSC line |
(Wen et al., 2014) |
https://doi.org/10.1038/nature13716
|
| Human: iPSC METTL8−/− (3 clones) |
This study |
N/A |
| Human: iPSC AAVS1 WT (4 clones) |
This study |
N/A |
| Human: HEK293 cell line |
ATCC |
Cat#CRL-1573 |
| Human: GP2-293 cell line |
Cellosaurus |
Cat#CVCL_WI48, RRID: CVCL_WI48 |
|
Experimental models: Organisms/strains
|
| Mouse: Mettl8flox/flox
|
This Study |
N/A |
| Mouse: B6.Cg-Tg(Nes-cre)1Kln/J |
The Jackson Laboratory |
JAX: 003771 |
| Mouse: Crl:CD1(ICR) |
Charles River Laboratory |
RRID: IMSR_CRL:22 |
| Mouse: C57BL/6 |
Charles River Laboratory |
RRID: IMSR_CRL:27 |
|
Oligonucleotides
|
| The primers and DNA oligos used for qPCR, cloning and genotyping |
See Table S1
|
N/A |
|
Recombinant DNA
|
| pCAGIG |
Addgene (Matsuda and Cepko, 2004) |
RRID:Addgene_11159 |
| pCAGIG/Mettl8-WT-Long |
This study |
N/A |
| pCAGIG/Mettl8-WT-Short |
This study |
N/A |
| pCAGIG/Mettl8-cKO-Long |
This study |
N/A |
| pCAGIG/Mettl8-cKO-Short |
This study |
N/A |
| pSpCas9(BB)-2A-Puro (PX459) V2.0 |
Addgene (Ran et al., 2013) |
RRID:Addgene_62988 |
| pSpCas9/METTL8-Upstream |
This study |
N/A |
| pSpCas9/METTL8-Downstream |
This study |
N/A |
| pSpCas9/AAVS1 |
This study |
N/A |
| Retro-X™ Universal Packaging System with pVSV-G |
Clontech |
Cat#631530 |
| pSUBGW vector |
(Zhang et al., 2016) |
N/A |
|
Software and algorithms
|
| Adobe Illustrator CC |
Adobe |
https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 |
| Adobe Photoshop CC |
Adobe |
https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 |
| Bcl2fastq v2.17.1.14 |
Illumina |
https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html; RRID: SCR_015058 |
| DESeq2 v1.36.0 |
(Love et al., 2014) |
https://bioconductor.org/packages/release/bioc/html/DESeq2.html; RRID:SCR_015687 |
| FlowJo |
FlowJo |
https://www.flowjo.com/solutions/flowjo; RRID: SCR_008520 |
| GraphPad Prism |
GraphPad Software |
https://www.graphpad.com/scientific-software/prism/; RRID: SCR_002798 |
| ImageJ |
NIH |
https://imagej.nih.gov/ij/; RRID: SCR_003070 |
| Imaris |
Bitplane |
https://imaris.oxinst.com/packages;RRID:SCR_007370 |
| Metascape |
(Zhou et al., 2019) |
http://metascape.org/gp/index.html#/main/step1; RRID: SCR_016620 |
| PANTHER v15.0 |
(Mi et al., 2019) |
http://www.pantherdb.org/; RRID: SCR_004869 |
| R Project v3.6.0 |
Open source |
https://www.r-project.org/; RRID: SCR_001905 |
| RStudio v1.2.1335 |
Open source |
https://rstudio.com/; RRID: SCR_000432 |
| STAR v2.5.2a |
(Dobin et al., 2013) |
https://github.com/alexdobin/STAR; RRID: SCR_015899 |
| Trimmomatic v0.32 |
(Bolger et al., 2014) |
http://www.usadellab.org/cms/index.php?page=trimmomatic; RRID:SCR_011848 |
|
Other
|
| 4-20% Mini-Gel 15-well, |
BIO-RAD |
Cat#4561096 |
| 6-well ultra-low attachment culture plate |
Fisher Scientific |
Cat#7200601 |
| Amersham Imager 600 |
GE healthcare |
N/A |
| Charged microscope slides |
Fisher Scientific |
Cat#22-035813 |
| Corning 96-Well Clear Ultra Low Attachment Microplates |
Corning/NETA |
Cat#7201680 |
| Forma Steri-Cult CO2 Incubator |
Thermo Fisher Scientific |
3310 |
| LSR II Flow Cytometer |
BD Biosciences |
N/A |
| MaxQ CO2 Plus Shaker |
Thermo Fisher Scientific |
88881102 |
| NextSeq550 |
Illumina |
SY-415-1002 |
| PCR tube strips |
Emsco/Fisher |
Cat#AB0490 |
| Qubit 3 Fluorimeter |
Thermo Fisher Scientific |
Cat#Q33216
|
| Seahorse XFe96 Analyzer |
Agilent |
N/A |
| StepOnePlus Real-Time PCR |
Applied Biosystems |
4376592 |
| TB Syringe (26 G × 3/8 in, 1 ml) |
BD Biosciences |
Cat#309625 |
| Trans-Blot Turbo Mini 0.2 um PVDF Transfer Packs |
BIO-RAD |
Cat#1704156 |
| Scanning Confocal microscope |
Carl Zeiss |
Zeiss LSM 780 |
