KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
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| Antibodies | ||
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| Rabbit polyclonal anti AKT | Cell Signaling Technology | Cat# 9272; RRID:AB_329827; 1:1000 |
| Rabbit anti phospho-AKT (S473) | Cell Signaling Technology | Cat# 4060; RRID:AB_2315049; 1:2000 |
| Rabbit anti phospho-AKT (T308) | Cell Signaling Technology | Cat# 13038; RRID:AB_2629447; 1:1000 |
| Rabbit anti RAS | Cell Signaling Technology | Cat# 67648; RRID:AB_2910195; 1:1000 |
| Rabbit anti AceCS1 | Cell Signaling Technology | Cat# 3658; RRID:AB_2222710; 1:1000 |
| Mouse anti ACLY | Cell Signaling Technology | Cat# 13390; RRID:AB_2798203; 1:1000 |
| Rabbit polyclonal anti phospho-ACLY (S455) | Cell Signaling Technology | Cat# 4331; RRID:AB_2257987; 1:1000 |
| Mouse anti SREBP | Santa Cruz Biotechnology | Cat #sc-13551; RRID:AB_628282; 1:1000 |
| Rabbit anti RAPTOR | Cell Signaling Technology | Cat# 2280; RRID:AB_561245; 1:1000 |
| Rabbit anti ERK1/2 | Cell Signaling Technology | Cat# 4695; RRID:AB_390779; 1:2500 |
| Rabbit anti phospho-ERK1/2 (T202/Y204) | Cell Signaling Technology | Cat# 9101; RRID:AB_331646; 1:2500 |
| Rabbit anti ACC | Cell Signaling Technology | Cat# 4190; RRID:AB_10547752; 1:1000 |
| Rabbit anti phospho-ACC (S79) | Cell Signaling Technology | Cat# 11818; RRID:AB_2687505; 1:1000 |
| Rabbit anti Ras-G12D | Cell Signaling Technology | Cat# 14429; RRID:AB_2728748; 1:1000 |
| Rabbit anti Ras-G12V | Cell Signaling Technology | Cat# 14412; RRID:AB_2714031; 1:1000 |
| Rabbit anti mTor | Cell Signaling Technology | Cat#2983; RRID:AB_2105622; 1:1000 |
| Rabbit anti phospho-mTor (S2448) | Cell Signaling Technology | Cat#2971; RRID:AB_330970; 1:1000 |
| Rabbit anti S6 | Cell Signaling Technology | Cat#2217; RRID:AB_331355; 1:1000 |
| Rabbit anti phospho-S6 (S235/236) | Cell Signaling Technology | Cat#4857; RRID:AB_2181035; 1:1000 |
| Rabbit anti 4E-BP1 | Cell Signaling Technology | Cat#9452; RRID:AB_331692; 1:1000 |
| Rabbit anti phospho-4E-BP1 (S65) | Cell Signaling Technology | Cat#9451; RRID:AB_330947; 1:1000 |
| Rabbit anti-acetylated lysine | Invitrogen | Cat# MA5-33031; RRID:AB_2810124; 1:1000 |
| Rabbit polyclonal anti Lamin A/C | Cell Signaling Technology | Cat #2032; RRID:AB_2136278; 1:1000 |
| Rabbit anti GAPDH | Cell Signaling Technology | Cat# 2118; RRID:AB_561053; 1:2500 |
| Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) | Jackson Immunoresearch | Cat# 115-035-003; RRID:AB_10015289; 1:10000 |
| Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) | Jackson Immunoresearch | Cat# 115-036-003; RRID:AB_2338518; 1:10000 |
|
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| Bacterial and virus strains | ||
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| Stellar Competent Cells | Takara | Cat# 636763 |
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| Chemicals, peptides, and recombinant proteins | ||
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| DMSO | Fisher | Cat# BP231-100 |
| Acetate | ThermoScientific | Cat# 537462 |
| Glucose | Sigma | Cat# G7021 |
| [U-13C]glucose | Cambridge Isotope Laboratories | Cat# CLM-1396-1 |
| [1,2-13C]acetate | Cambridge Isotope Laboratories | Cat# CLM-440-1 |
| Trichloroacetic acid (w/v) | Sigma | Cat# T6399 |
| DMEM Medium | Gibco | Cat# 10566-016 |
| RPMI Medium | Gibco | Cat# 72400-047 |
| Fetal Bovine Serum | HyClone | Cat# SH30071.03 |
| Penicillin Streptomycin | Gibco | Cat# 15150-122 |
| ACSS2 inhibitor | Selleck | Cat# S8588 |
| Acetyl-CoA, lithium salt | Sigma-Aldrich | Cat# A2181 |
| 5-sulfosalicylic acid | Sigma-Aldrich | Cat# A2130 |
| LC/MS grade acetonitrile | Fisher Scientific | Cat# A998-1 |
| Formic acid | Fisher Scientific | Cat# A117-05AMP |
| Methanol | Fisher Scientific | Cat# 4581 |
| TRI reagent solution | Invitrogen | Cat# AM9738 |
| Chloroform | Sigma-Aldrich | Cat# 650498 |
| TrypLE Express Enzyme | Gibco | Cat# 12605010 |
| 250X Cholesterol | Gibco | Cat# 12531-018 |
| Protease inhibitor cocktail | Sigma-Aldrich | Cat# 11836170001 |
| Phosphatase inhibitor cocktail | Sigma-Aldrich | Cat# 78420 |
| Lipofectamine™ 3000 | Invitrogen | Cat# L3000001 |
| SpCas9 nuclease | Synthego | Cat SpCas9 |
| L-glutamine | Gibco | Cat# 25030081 |
| HEPES | Gibco | Cat# 15630130 |
| Insulin | Lonza | Cat# BE02-033E20 |
| Hydrocortisone | Stemcell | Cat# 74142 |
| Thioglycerol | Sigma Aldrich | Cat# M6145 |
| PMSF | Thermo Scientific | Cat# 36978 |
| Dithiothreitol (DTT) | Thermo Scientific | Cat# R0861 |
| RIPA Buffer | Sigma Aldrich | Cat# R0278 |
| 4X Laemmli Buffer | BioRad | Cat# 1610747 |
| 2-mercaptoethanol | Fisher Scientific | Cat# 21985023 |
| Non-fat milk | BioRad | Cat# 1706404 |
| Lys-C Mass Spec Grade | Promega | Cat# VA117A |
| Acetonitrile | Thermo Scientific Chemicals | Cat# 047138.M1 |
| dialyzed FBS | Gibco | Cat# A3382001 |
| Ammonium acetate | Sigma | Cat# 533004 |
| Lipofectamine 3000 | Invitrogen | Cat# L3000015 |
| Puromycin | Gibco | Cat# A1113803 |
| Polybrene | Sigma-Aldrich | Cat# TR-1003 |
| Trametinib | Selleck | Cat# S2673 |
| Hydroxypropyl methylcellulose | Thermo Scientific Chemicals | Cat# 044779.A1 |
| Tween-80 | Sigma Aldrich | Cat# P1754 |
| Polyethylene glycol 300 (PEG-300) | Selleck | Cat# S6704 |
|
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| Critical commercial assays | ||
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| Mycoplasma Detection Kit | ATCC | Cat# 30-1012K |
| TruSeq RNA Sample pre-kit-V2 | Illumina | Cat# RS-122-2001 |
| Cholesterol/Cholesterol Ester-Glo assay | Promega | Cat# J3190 |
| Active Ras Detection Kit | Cell Signaling Technology | Cat# 8821 |
| Pierce BCA Protein Assay Kit | Thermo Scientific | Cat# 23225 |
| CellTiter-Glo 2.0 | Promega | Cat# G9241 |
| NE-PER Nuclear and Cytoplasmic Extraction Kit | Thermo Scientific | Cat# 78833 |
|
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| Deposited data | ||
|
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| KRAS Transcriptomics | This study | Geo: GSE261333 |
| KRAS Proteomics | This study | Pride: PXD050157 |
|
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| Experimental models: Cell lines | ||
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| HEK293 | ATCC | Cat# CRL-1573; |
| LIM1215 KRASWT/WT | Sigma Aldrich | Cat# 10092301 |
| LIM1215 KrasWT/G12D | Horizon Discovery | Cat# HD 116-005 |
| LIM1215 KrasWT/G12V | Horizon Discovery | Cat# HD 116-006 |
| LIM1215 KrasWT/G13D | Horizon Discovery | Cat# HD 116-009 |
| SW48 KrasWT/WT | ATCC | Cat# CCL231 |
| SW48 KrasWT/G12D | Horizon Discovery | Cat# HD 103-011 |
| SW48 KrasWT/G12V | Horizon Discovery | Cat# HD 103-007 |
| CRC adenoma cells Apc−/−::KrasWT/G12D | K. Haigis | NA |
| CRC adenoma cells Apc−/−::KrasWT/G12V | Budagyan and Chernoff22 | NA |
| CRC adenoma cells Apc−/−::KrasWT/G12R | Budagyan and Chernoff22 | NA |
| CRC adenoma cells Apc−/−::KrasWT/G13D | Budagyan and Chernoff22 | NA |
|
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| Experimental models: Organisms/strains | ||
|
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| C57BL/6J | Jackson Laboratories | Cat# JAX:00664 |
|
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| Oligonucleotides | ||
|
| ||
| Acss2 sgRNA | Synthego | NA |
|
Kras forward primer 5′-GGGTAGGTGTTGGGATAGCTGTCGACAAGC-3′ |
Integrated DNA Technologies | NA |
|
Kras reverse primer 5′-CTTTACAAGCGCACGCAGACTGTAGAGC-3′ |
Integrated DNA Technologies | NA |
|
Kras sgRNA top primer 5′- CACCGTGGTTGGAGCTGATGGCGT-3′ |
Integrated DNA Technologies | NA |
|
Kras sgRNA bottom primer 5′-AAACACGCCATCAGCTCCAACCAC-3′ |
Integrated DNA Technologies | NA |
| BFP sgRNA top primer 5′- CACCGCACTGCACCCCGTGGCTCA-3′ |
Integrated DNA Technologies | NA |
| BFP sgRNA bottom primer 5′-AAACTGAGCCACGGGGTGCAGTGC-3′ |
Integrated DNA Technologies | NA |
| ssODN GFP 5′CACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC -3′ | Integrated DNA Technologies | NA |
| ssODN KrasG12V 5′TGCTGAAAATGACTGAGTATAAGCTTGTGGTGGTTGGCGCCGTTGGAGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCA-3′ |
Integrated DNA Technologies | NA |
| ssODN KrasG12R 5 TGCTGAAAATGACTGAGTATAAGCTTGTGGTGGTTGGCGCCGTGTGAGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCA-3′ |
Integrated DNA Technologies | NA |
| ssODN KrasG13D 5 TGCTGAAAATGACTGAGTATAAGCTTGTGGTGGTTGGCGCCGGTGATGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCA-3′ |
Integrated DNA Technologies | NA |
|
Kras sequencing primer 5′TCTTGTGTGAGACATG-3′ |
Integrated DNA Technologies | NA |
| Acss2 TaqMan Gene Expression Assay | Invitrogen | Cat# Mm00480101_m1 |
| Hmgcs1 TaqMan Gene Expression Assay | Invitrogen | Cat# Mm1304569_m1 |
| Srebf1 TaqMan Gene Expression Assay | Invitrogen | Cat# Mm00550338_m1 |
| Rplp0 TaqMan Gene Expression Assay | Invitrogen | Cat# Mm00725448_s1 |
|
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| Recombinant DNA | ||
|
| ||
| pLentiCas9-P2A-BFP | Addgene | RRID:Addgene_149447 |
| U6:sgBFP-U6:sgKrasG12D-pX333-HC-Red | Addgene | RRID:Addgene_149448 |
| pLJMI-ACSS2 | J.C. Alwine | Vysochan et al.53 |
| pTRIPZ | Addgene | RRID:Addgene_206981 |
| pTRIPZ shACSS2.1 | K. Rathmell | Bacigalupa et al.54 |
| pTRIPZ shACSS2.2 | K. Rathmell | Bacigalupa et al.54 |
|
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| Software and algorithms | ||
|
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| GraphPad Prism | GraphPad Software, Inc. | www.graphpad.com/ |
| Adobe Illustrator | Adobe Creative Cloud | https://www.adobe.com/creativecloud.html |
| Proteome Discoverer 2.2 | Thermo Fisher Scientific | https://www.thermofisher.com/order/catalog/product/OPTION-30795 |
| R Framework | Team RCR: A Language and Environment for Statistical Computing | http://www.R-project.org/ |
| Python 3.7.3 | Python Software Foundation | https://www.python.org/ |
| Bio Render App | Bio Render | https://www.biorender.com/ |
| XCalibur 4.0 | Thermo Scientific | OPTON-30965 |
| Tracefinder 5.1 | Thermo Scientific | OPTON-31001 |
| Cas-OFFinder | CRISPR RGEN Tools | http://www.rgenome.net/cas-offinder |
| IDT CRISPR | Integrated DNA Technologies | https://www.idtdna.com/page/products/crispr-genome-editing |
| Xcelligence RTCA software | Agilent | www.agilent.com |
| Synergy Finder+ web application | SynergyFinder | www.synergyfinder.org |
| Tophat2 | Johns Hopkins University/Github | https://github.com/infphilo/tophat |
| Cufflinks | Github | https://github.com/cole-trapnell-lab/cufflinks |