Single-cell heterogeneity of EGFR and CDK4 co-amplification is linked to immune infiltration in glioblastoma

. Author manuscript; available in PMC: 2023 Apr 19.

Published in final edited form as: Cell Rep. 2023 Mar 14;42(3):112235. doi: 10.1016/j.celrep.2023.112235

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal anti-CD163 (clone EPR19518)	Abcam	Cat#ab182422; RRID:AB_2753196
Goat anti-rabbit Alexa Fluor 568	Invitrogen	Cat#A11011; RRID:AB_143157
Cd45-PE-Cy7	BD Biosciences	Cat#561868; RRID:AB_10893599
Cd11b-AlexaFluor700	BD Biosciences	Cat#557960; RRID:AB_396960
Ly6C-PerCP-Cy5.5	BD Biosciences	Cat#560525; RRID:AB_1727558
Ly6G-PacificBlue	Biolegend	Cat#127612; RRID:AB_2251161
anti-Egfr	Cell Signaling	Cat#2232S
anti-Cdk4	Abcam	Cat#ab137675
anti-GAPDH	Cell Signaling	Cat#8884S
Biological samples
GBM FFPE blocks	Mount Sinai Medical Center, Miami, Florida, USA	N/A
Chemicals, peptides, and recombinant proteins
dUTP	Abbott Molecular	Cat#02N32-050
Human COT1 DNA	Invitrogen	Cat#15279011
Vysis CEP hybridization buffer	Abbott Molecular	Cat#07J36-001
UltraPure 20x SSC	Invitrogen	Cat#15557044
NP-40	Sigma	Cat#13021
ProLong Gold Antifade mountant with DAPI	Invitrogen	Cat#P36931
dNTP	New England Bio Labs	Cat#N0446
7-deaza- 2′-deoxy-GTP	Roche	Cat#10988537001
Betaine	Alfa Aesar	Cat#J77507VCR
Platinum Taq Polymerase	Life Technologies	Cat#10966083
Proteinase K	ThermoFisher	Cat#AM2548
Antigen retrieval solution pH9	Dako	Cat#S2367
Goat serum	ThermoFisher	Cat#31873
TruStain FcX PLUS	Biolegend	Cat#156603
Staining Buffer	Biolegend	Cat#420201
Critical commercial assays
QiaAmp DNA FFPE Tissue Kit	Qiagen	Cat#56404
AmpliSeq Cancer HotSpot Panel v2 kit	Illumina	Cat#20019161
GeoMx Digital Spatial Profiler (DSP) Protein panel - service	Nanostring	N/A
Nick Translation Kit	Abbott	Cat#07J00-001
DNA PCR-Free Library Prep kit	Illumina	Cat# 20041795
Deposited data
Whole genome sequencing	This paper	phs003100.v1.p1
Single-cell RNA sequencing from GEO	Neftel et al.¹⁵	GSE131928
Single-cell RNA sequencing from Broad Institute Single-Cell Portal	Richards et al.¹⁶	SCP503
Experimental models: Cell lines
U-87 MG	ATTC	Cat#HTB-14
DBTRG	DSMZ	Cat#ACC 359
HEK-293T	Sigma	Cat#12022001
GL261	DSMZ	Cat#ACC802
Oligonucleotides
STAR-FISH hTERT F1: 5′CTATGGTTCCAGGCCCGTTC-3′	This paper	N/A
STAR-FISH hTERT R1: 5′GGCTCCCAGTGGATTCGC-3′	This paper	N/A
STAR-FISH hTERT F2: 5′TGTCGACGCAAAACCGGTTCCGGCCCAGCCCTTT-3′	This paper	N/A
STAR-FISH hTERT R2: 5′GCGATATGACGACGCGAATACCCACGTGCGCAGC-3′	This paper	N/A
Specific-to-allele PCR-FISH: +T + G+TCGACGCAAAACCGG+T+T + C (+ indicates LNA modified bases)	This paper	N/A
Recombinant DNA
BAC clone RP11-339F13 (EGFR gene)	BACPAC Genomics	N/A
BAC clone RP11-231C18 (PDGFRA gene)	BACPAC Genomics	N/A
BAC clone RP11-571M6 (CDK4 gene)	BACPAC Genomics	N/A
pLenti-CMV-Puro	Campeau et al.⁶³	Addgene Plasmid #17452
pLenti-CMV-Neo	Campeau et al.⁶³	Addgene Plasmid #17392
pLV-eGFP	Enomoto et al.⁶⁴	Addgene Plasmid #36083
pLV-mCherry	Gift from Pantelis Tsoulfas	Addgene Plasmid #36084
pLenti-CMV-Egfr-Puro	This paper	N/A
pLenti-CMV-Cdk4-Neo	This paper	N/A
Software and algorithms
ImageJ	Schneider et al.⁶⁵	https://imagej.nih.gov/ij/
R software	R Core Team⁶⁶	https://www.r-project.org/
Python	Rossum et al.⁶⁷	https://www.python.org/
Single cell FISH counting macro for ImageJ	Janiszewska et al.⁶⁸	https://doi.org/10.1172/jci.insight.147617
FlowJo	Beckman Dickinson	https://www.flowjo.com/
AmpliconArchitect	Deshpande et al.³⁸	https://github.com/nf-core/circdna.git
Code related to analyses	This paper	https://github.com/Michorlab/GBM_OR_immune https://doi.org/10.5281/zendo.7618100