Key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | mCherry (Rabbit poly-clonal) | abcam | ab167453 | IF (1 in 500) |
| Antibody | Rpb1 NTD (D8L4Y) (Rabbit mono-clonal) | Cell Signaling Technology | #14958 | IF (1 in 1000) |
| Antibody | Anti-Digoxigenin (Sheep poly-clonal) | Roche | Ref 11333089001 | DNA FISH (1 in 10) |
| Antibody | Secondary Antibody – Alexa Fluor 647 (Donkey anti-Sheep IgG poly-clonal) | Thermo Fisher Scientific | A-21448 | DNA FISH (1 in 10) |
| Antibody | Secondary Antibody – Alexa Fluor 568 (Donkey anti-Rabbit IgG poly-clonal) | Thermo Fisher Scientific | A-10042 | IF (1 in 1000) |
| Antibody | Secondary Antibody – Alexa Fluor 488 (Donkey anti-Rabbit IgG poly-clonal) | Thermo Fisher Scientific | A-21206 | IF (1 in 1000) |
| Antibody | Secondary Antibody – Alexa Fluor 488 (Donkey anti-Rat IgG poly-clonal) | Thermo Fisher Scientific | A-21208 | IF (1 in 1000) |
| Genetic reagent (human) | Fosmid FISH probe (Human) | BACPAC resource | https://bacpacresources.org/library.php?id=275 | See Materials and methods - Supplementary file 1 |
| Cell line (Homo sapiens) | E20, E25, E26, E28, NSC – GCGR Human Glioma Stem Cells | This paper, Glioma Cellular Genetics Resource, CRUK, UK | http://gcgr.org.uk; pending publication | |
| Other | DMEM/HAMS-F12 | Sigma-Aldrich | Cat#: D8437 | Cell culture, media |
| Chemical compound, drug | Pen/Strep | GIBCO | Cat#: 15140–122 | Cell culture, media supplement |
| Other | BSA Solution | GIBCO | Cat#: 15260–037 | Cell culture, media supplement |
| Other | B27 Supplement (×50) | LifeTech/GIBCO | Cat#: 17504–044 | Cell culture, media supplement |
| Other | N2 Supplement (×100) | LifeTech/GIBCO | Cat#: 17502–048 | Cell culture, media supplement |
| Other | Laminin | Cultrex | Cat#: 3446-005-01 | Cell culture, media supplement, and pre-lamination of culture vessels |
| Peptide, recombinant protein | EGF | Peprotech | Cat: 315–09 | Cell culture, media supplement |
| Peptide, recombinant protein | FGF-2 | Peprotech | 100-18B | Cell culture, media supplement |
| Other | Accutase | Sigma-Aldrich | Cat#: A6964 | Cell culture, cell dissociation agent |
| Other | DMSO | Sigma-Aldrich | Cat#: 276855 | Cell culture, freeze media, and drug diluent |
| Other | Triton X-100 | Merck Life Sciences | Cat#: X-100 | Cell permeabiliz-ation agent following cell fixation |
| Other | Paraformaldehyde Powder 95% | Sigma | Cat#: 158127 | Cell fixation agent |
| Other | Tween 20 | Cambridge Bioscience | Cat#: TW0020 | DNA FISH (hybridization mix) |
| Other | PBS Tablets | Sigma-Aldrich | Cat#: P4417 | Diluent and washing agent |
| Other | Ethanol | VWR | Cat#: 20821–330 | DNA FISH |
| Other | Methanol | Fisher Chemical | M/4000/17 | Used 3:1 with acetic acid for metaphase spreads |
| Other | Acetic acid | Honeywell Research Chemicals | 33209-1L | See above |
| Peptide, recombinant protein | Alexa Fluor 647 EGF complex | Thermo Fisher Scientific | E35351 | Flow cytometry |
| Other | Green496-dUTP | ENZO Life Sciences | ENZ-42831L | Direct labelling of Fosmid DNA FISH probes via nick translation |
| Other | ChromaTide Alexa Fluor 594–5-dUTP | Thermo Fisher Scientific | C11400 | Direct labelling of Fosmid DNA FISH probes via nick translation |
| Peptide, recombinant protein | DNA Polymerase 1 | Invitrogen | 18010–017 | |
| Peptide, recombinant protein | DNase I recombinant, RNase-free | Roche | 04716728001 | |
| Genetic reagent (human) | Human Cot-1 DNA | Thermo Fisher Scientific | 15279011 | |
| Genetic reagent (salmon) | Salmon Sperm DNA | Invitrogen | 15632011 | |
| Chemical compound, drug | Paclitaxel | Cambridge Bioscience | CAY10461 | 10–100 nM |
| Chemical compound, drug | Nocodazole | Sigma-Aldrich | SML1665 | 50–100 ng/ml |
| Other | XCP 7 Orange Chromosome Paint | MetaSystems Probes | D-0307-100-OR | DNA FISH (see Figure 1 and Materials and methods referring to this) |
| Commercial assay or kit | Stellaris RNA-FISH probes (Custom Assay with Quasar 570 Dye) | LGC Biosearch Technologies | SMF-1063–5 | RNA FISH |
| Commercial assay or kit | Stellaris RNA FISH Hybridization Buffer | LGC Biosearch Technologies | SMF-HB1-10 | RNA FISH |
| Genetic reagent (human) | Alt-R CRISPR-Cas9 crRNA | IDT-Technologies | Alt-R CRISPR-Cas9 crRNA | |
| Genetic reagent (human) | Alt-R CRISPR-Cas9 tracrRNA | IDT-Technologies | 1072532 | |
| Commercial assay or kit | SG Cell Line 4D-NucleofectorTM X Kit S | Lonza Bioscience | V4XC-3032 | |
| Genetic reagent (human) | Chromosome 7 Control Probe | Pisces Scientific | CHR07-10-DIG | Probe and hybridization mix |
| Other | DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | Nuclear staining; 50 ng/ml and 5 ng/ml (as indicated in Materials and methods) |
| Sequence-based reagent | mCherry_PolR2G crRNA and dsDNA (donor) | Twist Bioscience | See Materials and methods and Supplementary file 1 | |
| Other | WGS and RNAseq | This paper Glioma Cellular Genetics Resource, CRUK, UK |
GEO: GSE215420 See also: https://gcgr.org.uk |
See Materials and methods |
| Other | Erosion Territories analysis | This paper | Code available at: https://github.com/IGC-Advanced-Imaging-Resource/Purshouse2022_paper | |
| Other | Cluster analysis | This paper | Code available at: https://github.com/SjoerdVBeentjes/ripleyk | |
| Other | RNA-seq/WGS analysis | This paper | Code available at: https://github.com/kpurshouse/ecDNAcluster | |
| Software, algorithm | GraphPad Prism 9.0 | GraphPad Software, Inc | https://www.graphpad.com/ | |
| Software, algorithm | FCS Express | FCS Express 7 | https://denovosoftware.com/ | |
| Software, algorithm | Fiji/ImageJ | Open Source | https://imagej.net/Fiji | |
| Software, algorithm | BioRender | BioRender | https://biorender.com/ | |
| Software, algorithm | Python v3.9 | Open Source | https://www.python.org | |
| Software, algorithm | Algorithm - RipleyK package | Python Package Index | https://pypi.org/project/ripleyk/ | |
| Software, algorithm | Imaris x64 v9.4.0 | Imaris Microscopy Image Analysis Software | https://imaris.oxinst.com/ | |
| Software, algorithm | UCSC Genome Browser | Kent et al., 2002 | https://genome.cshlp.org/content/12/6/996 | |
| Software, algorithm | STAR 2.7.1a | Dobin et al., 2013 | https://github.com/alexdobin/STAR; Dobin et al., 2013 | |
| Software, algorithm | Picard | Broad Institute |
https://broadinstitute.github.io/picard/ RRID:SCR_006525, Version 2.23.2 |
|
| Software, algorithm | AmpliconArchitect | Deshpande et al., 2019 | https://github.com/virajbdeshpande/AmpliconArchitect; Deshpande et al., 2019 (with Python v2.7) | |
| Software, algorithm | AmpliconClassifier | Kim et al., 2020 | https://github.com/jluebeck/AmpliconClassifier (with Python v2.7) | |
| Software, algorithm | deepTools v3.4 | Ramírez et al., 2016 | https://deeptools.readthedocs.io/en/develop/ | |
| Software, algorithm | HOMER2 4.10 | Heinz et al., 2010 | http://homer.ucsd.edu/homer/ | |
| Software, algorithm | SAMtools v1.10 | Li et al., 2009 | http://www.htslib.org | |
| Software, algorithm | BEDTools v2.3 | Quinlan and Hall, 2010 | http://code.google.com/p/bedtools | |
| Software, algorithm | bcftools | Danecek et al., 2021 | https://doi.org/10.1093/gigascience/giab008 | |
| Software, algorithm | strelka v2.9.10 | Kim et al., 2018 | https://doi.org/10.1038/s41592-018-0051-x |