SON is an Essential m6A Target for Hematopoietic Stem Cell Fate

. Author manuscript; available in PMC: 2024 Dec 7.

Published in final edited form as: Cell Stem Cell. 2023 Dec 7;30(12):1658–1673.e10. doi: 10.1016/j.stem.2023.11.006

KEY RESOUCRCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD45.1-PE TexasRed	BD Bioscience	Cat# 562452, RRID:AB_11152958
CD45.2-A700	Invitrogen	Cat# 56-0454-82, RRID:AB_657752
CD3-PECy5	Invitrogen	Cat# 15-0031-83, RRID:AB_468691
CD8-PECy5	Invitrogen	Cat# 15-0081-83, RRID:AB_468707
Gr1-PECy5	Invitrogen	Cat# 15-5931-82, RRID:AB_468813
B220-PECy5	Invitrogen	Cat# 15-0452-83, RRID:AB_468756
CD19-PECy5	Invitrogen	Cat# 15-0193-83, RRID:AB_657673
Ter119-PECy5	Invitrogen	Cat# 15-5921-82, RRID:AB_468810
cKit-APC-Cy7	BioLegend	Cat# 105826, RRID:AB_1626278
Sca-1-Pacific Blue	BioLegend	Cat# 122520, RRID:AB_2143237
CD150-APC	BioLegend	Cat# 115910, RRID:AB_493460
CD48-PE	BD Bioscience	Cat# 557485, RRID:AB_396725
Gr1-APC	Invitrogen	Cat# 17-5931-82, RRID:AB_469476
Mac1-Pacific Blue	BioLegend	Cat# 101224, RRID:AB_755986
Ter119-PE-Cy5	Invitrogen	Cat# 15-5921-82, RRID:AB_468810
CD41-PE	BD Bioscience	Cat# 558040, RRID:AB_397004
CD3-Pacific Blue	eBioscience	Cat# 48-0031-80, RRID:AB_10733280
CD4-PE	BD Bioscience	Cat# 557308, RRID:AB_396634
B220-PE-Cy7	BD Bioscience	Cat# 552772, RRID:AB_394458
CD19-PE-Cy5	Invitrogen	Cat# 15-0193-83, RRID:AB_657673
IgM-APC	BioLegend	Cat# 406509, RRID:AB_315059
Sca-1-PECy7	BioLegend	Cat# 122514, RRID:AB_756199
CD48-FITC	BioLegend	Cat# 103404, RRID:AB_313019
human CD45 PerCP Cy5.5	eBioscience	Cat# 45-0459-42, RRID:AB_10717530
mouse CD45 APC	BD Bioscience	Cat# 559864, RRID:AB_398672
anti-SON	Abcam	Cat# ab121759, RRID:AB_11132447
anti-CCL5	thermo	Cat# 710001, RRID:AB_2532515
Anti-METTL3	Proteintech	Cat# 150731-1-AP, RRID: AB_2142033
Anti-ACTIN	Sigma	Cat# A3854, RRID: AB_262011
Anti-YTHDF1	Abcam	Cat# ab220162, RRID: AB_2892231
Anti-YTHDF2	Proteintech	Cat# 24744-1-AP, RRID: AB_2687435
Anti-YTHDF3	Abcam	Cat# A3854, RRID: AB_2868574
Anti-VINCULIN	NovusBio	Cat# NBP2-20859, RRID: N/A
donkey anti-mouse Alexa fluor 568	Invitrogen	Cat# A-10037, RRID: AB_2534013
donkey anti-rabbit Alexa fluor 647	Invitrogen	Cat# A-31573, RRID:AB_2536183
dsRNA-specific antibody J2	Abcam	Cat# ab288755
streptavidin-Cy5	Invitrogen	Cat# 43-4316
anti-NUMB	Abcam	Cat# ab4147, RRID:AB_304320
anti-MYC	Cell Signaling	Cat# 5605, RRID:AB_1903938
donkey anti-goat Alexa fluor 488	Invitrogen	Cat# A-11055, RRID:AB_2534102
donkey anti-goat Alexa fluor 647	Invitrogen	Cat# A-21447, RRID:AB_141844
donkey anti-rabbit Alexa fluor 568	Invitrogen	Cat# A10042, RRID:AB_2534017
Anti-mouse IgG	Sigma	Cat# 12-371, RRID: AB_145840
anti-Flag antibody	Sigma-Aldrich	Cat# F1804, RRID:AB_262044
Bacterial and virus strains
N/A	N/A	N/A
Biological samples
Cord blood	National Cord Blood Program, NY Blood Center	N/A
Chemicals, peptides, and recombinant proteins
pIpC HMW	InVivogen	Cat# Vac-Pic
Pyronin Y	Sigma	Cat# 213519
Polybrene	Millipore	Cat# TR-1003-G
Murine SCF	Peprotech	Cat# 250-03
Murine IL-3	Peprotech	Cat# 213-13
Murine IL-6	Peprotech	Cat# 216-16
Mouse TPO	Peprotech	Cat# 315-14
Mouse Flt3L	Peprotech	Cat# 250-31L
SFEM medium	Stem Cell Technologies	Cat# NC9753895
X-vivo 10 medium	Lonza	Cat# BE04-380Q
Human SCF	Peprotech	Cat# 300-07
Human Flt3L	Peprotech	Cat# 300-19
Human TPO	Peprotech	Cat# 300-18
Human IL7	Peprotech	Cat# 200-07
STM2457	MedChemExpress	Cat# 134836
Recombinant mouse CCL5	R&D Systems	Cat# 478-MR-025
Critical commercial assays
Murine CD117 MicroBeads Kit	Miltenyi Biotec	Cat# 130-091-224
Verso cDNA Synthesis Kit	Thermo Fisher Scientific	Cat# AB1453-B
Power SYBR Green PCR Master Mix	Thermo Fisher Scientific	Cat# 4367659
Human CD34 MicroBeads Kit	Miltenyi Biotec	Cat# 130-046-702
Dynabeads Protein A/G	Invitrogen	Cat# 10015D
M.O.M (Mouse on Mouse) Immunodetection kit	Vector Laboratories	Cat# BMK-2202
Neon transfection Kit	Invitrogen	Cat# N-1096
Click-iT Nascent RNA Capture Kit	Thermo Fisher Scientific	Cat# C10365
Deposited data
Mouse HSC and MPP DART-seq data	This paper	GSE231862
Human CD34 HSPC DART-seq data	This paper	GSE231861
Single cell RNA-seq data	This paper	GSE231863
Bulk RNA-seq data	This paper	GSE231637
Experimental models: Cell lines
Human: THP1 leukemia cell line	ATCC	TIB-202
Experimental models: Organisms/strains
Mouse: Mx1-cre Mettl3 flox/flox	Cheng et al¹⁸	N/A
Mouse: Gt(ROSA)26Sortm1.1(CAG-cas9,-EGFP)Fezh/J (Rosa*CAS9)	The Jackson Laboratory	Strain #024858
Mouse: RosaCAS9 Mx1-cre Mettl3 flox/flox	This paper	N/A
Mouse: C57BL/6J	The Jackson Laboratory	Strain # 000664
Mouse: NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ	The Jackson Laboratory	Strain # 013062
Mouse: B6.SJL-Ptprc <a> /Boy	Taconic Biosciences	Strain # 4007
Oligonucleotides
shCCL5-1 oligo F: CCGGTCTTGATTCTGACCCTGTATACTCGAGTATACAGGGTCAGAATCAAGATTTTTG	This paper	N/A
shCCL5-1 oligo R: AATTCAAAAATCTTGATTCTGACCCTGTATACTCGAGTATACAGGGTCAGAATCAAGA	This paper	N/A
shCCL5-2 oligo F: CCGGCCACGTCAAGGAGTATTTCTACTCGAGTAGAAATACTCCTTGACGTGGTTTTTG	This paper	N/A
shCCL5-2 oligo R: AATTCAAAAACCACGTCAAGGAGTATTTCTACTCGAGTAGAAATACTCCTTGACGTGG	This paper	N/A
sgSON-1: cctgaaaacctgctcgatgt	This paper	N/A
sgSON-4: gggtcttagatacggagctg	This paper	N/A
sgCCL5-3: tgcagagggcggctgcagtg	This paper	N/A
sgCCL5-4: gtgaactaagtacctaccgt	This paper	N/A
Primers, see Supplementary Table S10	This paper	N/A
Targeted Son demethylation oligoes, see Supplementary Table S10	This paper	N/A
sgRNAs against mus YTHDF1/2/3, see Supplementary Table S10	This paper	N/A
Recombinant DNA
Lenti-EGFP-Blast	This paper	N/A
Lenti-SON-EGFP-Blast	This paper	N/A
MSCV-IRES-GFP	Addgene	# 20672
MSCV-APOBEC-YTH	This paper	N/A
MSCV-SON-N-GFP	This paper	N/A
MSCV-SON-C-GFP	This paper	N/A
MSCV-SON-delDB-GFP	This paper	N/A
MSCV-SON-RB-GFP	This paper	N/A
MSCV-SON-DB-GFP	This paper	N/A
MSCV-SON-SRRB-GFP	This paper	N/A
FTO-dCas9	Addgene	# 134781
PLKO.1.GFP	This paper	N/A
PLKO5.sgRNA.EFS.GFP	Addgene	# 57822
Software and algorithms
Image J version 2.1.0	Image J	N/A
FlowJo software (version10.2)	FlowJo	N/A
GraphPad Prism 9	GraphPad Software	N/A
Imaris	BITPLANE	N/A
STAR	Dobrin et al.⁸⁷	https://github.com/alexdobin/STAR
GATK	Broad institute	https://gatk.broadinstitute.org/hc/en-us/articles/360035531192
HOMER (version 4.11)	Heinz et al.⁸⁸	http://homer.ucsd.edu/homer/
HISAT3N (version 2.2.1-3n)	Zhang et al.⁷⁸	http://daehwankimlab.github.io/hisat2/hisat-3n/
Bullseye	Tegowski et al.⁸⁹	https://github.com/mflamand/Bullseye
MEME suite (version 1.4.1)	Bailey et al.⁷⁹	https://meme-suite.org/meme/
Cutadapt (version 4.1)	Marcel Martin	https://github.com/marcelm/cutadapt
HTSeq (version 2.0.2)	Putri et al.⁹⁰	https://github.com/htseq/htseq
DESeq (version 1.34.0)	Love et al.⁹¹	https://github.com/mikelove/DESeq2
EnhancedVolcano (version 1.12.0)	Blighe et al	https://github.com/kevinblighe/EnhancedVolcano
VAST-TOOLS (version 2.5.1)	Irimia et al⁹²	https://github.com/vastgroup/vast-tools
gplots (version 3.1.3)	T. Galili	https://github.com/talgalili/gplots
Cell Ranger (version 7.1.0)	Zheng et al.⁹³	N/A
Seurat (version 4.3.0)	Stuart et al.⁹⁴	https://github.com/satijalab/seurat
Harmony (version 0.1.1)	Korsunsky et al.⁸²	https://github.com/immunogenomics/harmony
clustree (version 0.5.0)	Oshlack et al.⁸³	https://github.com/lazappi/clustree
SCINA (version 1.2.0)	Zhang et al.⁹⁵	https://github.com/jcao89757/SCINA
scDissector	Martin et al.⁸⁶	https://github.com/effiken/scDissector