Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

. 2023 Mar 7;4(3):100962. doi: 10.1016/j.xcrm.2023.100962

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

CD45 BB515 (Clone HI30)	BD	Cat# 564586; RRID: AB_2869588
CD38 PE-Cy7	BD	Cat# 335790; RRID: AB_399969
CD45 APC	Invitrogen	Cat# MHCD4505; RRID: AB_10372216
CD34 BV421 (Clone 581)	BD	Cat# 562577; RRID: AB_2687922
CD8-PE-Cy 5.5	BD	Cat# 555368; RRID: AB_395771
CD56⁻ PE-Cy 5.5	BD	Cat# 555517; RRID: AB_395907
CD4⁻ PE-Cy 5.5	BD	Cat# 555348; RRID: AB_395753
CD3⁻ PE-Cy 5.5	BD	Cat# 555334; RRID: AB_395741
CD19⁻ PE-Cy 5.5	BD	Cat# 555414; RRID: AB_395814
CD2⁻ PE-Cy 5.5	BD	Cat# 555328; RRID: AB_395735
CD14⁻ PerCP-Cy5.5	BD	Cat# 550787; RRID: AB_393884
CD8-PE-Cy 5.5	BD	Cat# 555368; RRID: AB_395771
RBFOX2 (anti-Fox2/RBM9)	Abcam	Cat# ab57154; RRID: AB_2285090
Goat anti-mouse IgG2a H&L (PE)	Abcam	Cat# ab74490; RRID: AB_1310345

Bacterial and virus strains

2^ND and 3^rd Generation HIV Lentivirus	Core facility at UC San Diego	N/A

Biological samples

CD34⁺ Cord Blood	AllCells or Lonza	https://allcells.com; Cat# 2C-101
Adult AML patient samples	Obtained from patients in clinical trials of the Dutch-Belgian Cooperation Trial Group for Hematology-Oncology (HOVON) according to METc-VUmc	N/A
Adult secondary AML patient samples	Obtained through patients consented at UC San Diego Health according to Institutional Review Board-approved protocols	For patient characteristics see Crews et al.⁴
Pediatric AML and pediatric non-leukemic patient samples	Obtained from patients through a written informed consent from the Dutch Childhood Oncology Group (DCOG) according to METc-VUmc	N/A

Chemicals, peptides, and recombinant proteins

Rebecsinib (17S-FD-895)	Dr. Michael Burkart (Villa et al.⁵⁵)	N/A

Critical commercial assays

RNeasy Micro Kit	Qiagen	Cat# 74004
Super-Script III	ThermoFisher Scientific	Cat# 11752250
SYBR GreenER	ThermoFisher Scientific	Cat# 11761500
Live/Dead Fixable NearIR Dead Cell Stain Kit	ThermoFisher Scientific	Cat# L10119
Live/Dead Fixable Aqua Dead Cell Stain Kit	ThermoFisher Scientific	Cat# L34957
CD34 Micro-Bead Kit, Ultra-Pure, human	Miltenyi	Cat# 130-100-453
MyeloCult™ H5100	Stem Cell Technologies	Cat# 05150
MethoCult™ H4330	Stem Cell Technologies	Cat# 04330
Mission Bio Tapestri Single Cell (Myeloid DNA Core Ambient Kit Version 2)	Mission Bio	https://missionbio.com

Deposited data

Analyzed data and code	This paper	https://github.com/ucsd-ccbb/pAML_StemCell_Methods
Generated dataset	This paper	dbGaP: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs003196.v1.p1; accession code: phs003196.v1.p1

Experimental models: Cell lines

Kasumi-1	ATCC	Cat# CRL-2724; RRID:CVCL_0589
THP-1	ATCC	Cat# TIB-202; RRID:CVCL_0006
KG1a	ATCC	Cat# CCL-246.1; RRID:CVCL_1824
MOLM-13	DMSZ	Cat# ACC-554; RRID:CVCL_2119
SI/SI (IL-3, CSF) (SL)	StemCell Technologies Ltd.	Cat# 00302
M2-10B4 (IL-3, G-CSF) (M2)	StemCell Technologies Ltd.	Cat# 00301

Experimental models: Organisms/strains

Mouse: NOD.Cg-Prkdc^scidIl2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS-SGM)	Jackson Labs	N/A

Oligonucleotides

Human HPRT primers	Crews et al.⁴	N/A
Human MCL1-L primers	Goff et al.⁵⁶	N/A
Human MCL1-S primers	Goff et al.⁵⁶	N/A

Recombinant DNA

MAPT Reporter	Stoilov et al.³⁵	N/A
pCDH-EF1a-IRES-Puro MAPT	This paper	See Figure 4
pSMART hEF1a ShRBFOX2	Horizon Discovery (Waterbeach, UK)	https://horizondiscovery.com/en/gene-modulation/knockdown/shrna/products/smartvector-lentiviral-shrna?nodeid=entrezgene-23543
pSMART hEF1a ShControl	Horizon Discovery (Waterbeach, UK)	https://horizondiscovery.com/en/gene-modulation/knockdown/controls/products/smartvector-lentiviral-controls

Software and algorithms

Bcl2fastq (v2.17)		https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html
FastQC	Wingett and Andrews⁵⁷	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
STAR	Dobin et al.⁵⁸	https://github.com/alexdobin/STAR
RSEM	Li and Dewey⁵⁹	https://deweylab.github.io/RSEM/
GENCODE		https://www.gencodegenes.org/human/release_19.html
EdgeR	Robinson et al.⁶⁰	http://bioconductor.org/packages/release/bioc/html/edgeR.html
Limma	Ritchie et al.⁶¹	https://www.bioconductor.org/packages/release/bioc/html/limma.html
Limma-voom	Law et al.⁶²	https://www.bioconductor.org/packages/release/bioc/html/limma.html
webGestalt	Liao et al.⁶³	http://www.webgestalt.org/
GSVA	Hänzelmann et al.⁶⁴	https://bioconductor.org/packages/release/bioc/html/GSVA.html
gProfiler	Reimand et al.⁶⁵	https://biit.cs.ut.ee/gprofiler/page/r
rMATS turbo	Shen et al.⁶⁶	https://github.com/Xinglab/rmats-turbo
rmats2sashimiplot		https://github.com/Xinglab/rmats2sashimiplot/
rMAPS2 Motifi Map	Hwang et al.⁴⁶	http://rmaps.cecsresearch.org/MTool/
Mosaic		https://github.com/MissionBio/mosaic-jupyter
STRING high confidence interactome	Szklarczyk et al.⁴⁷	9606.protein.links.v11.0.txt
Cytoscape	Shannon et al.⁶⁷	https://cytoscape.org/
VisJS2jupyter	Rosenthal et al.⁶⁸	https://github.com/ucsd-ccbb/visJS2jupyter
network_bio_toolkit		https://github.com/ucsd-ccbb/network_bio_toolkit
PicardTools		https://broadinstitute.github.io/picard/
Genome Analysis Toolkit (GATK)	McKenna et al.⁶⁹	https://gatk.broadinstitute.org/hc/en-us
RepeatMasker		https://www.repeatmasker.org/
FlowJo	FLOW JO LLC	https://www.flowjo.com/
GraphPad Prism	GraphPad Software Inc.	https://www.graphpad.com/scientific-software/prism/
Microsoft Excel	Microsoft
GATK Resource Bundle		https://gatk.broadinstitute.org/hc/en-us/articles/360035890811-Resource-bundle
Gnomad		https://gnomad.broadinstitute.org/

Other

Spliceosome Protein Annotations	Cvitkovic and Jurica⁷⁰	http://spliceosomedb.ucsc.edu/
ExAC		https://gnomad.broadinstitute.org/downloads#exac-variants
RNA Binding Protein Targets by eCLIP	Van Nostrand et al.³⁹	N/A
oRNAment	Benoit et al.⁴¹	N/A
REDIportal.80	Lo Giudice et al.⁷¹	N/A
dbSNP	Sherry et al.⁷²	N/A