Appendix 1—key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | OE19 | ACACC | 96071721 | |
| Cell line (H. sapiens) | CP-A | ATCC |
KR-42421 |
|
| Cell line (H. sapiens) | OE19-dCas9-KRAB | This study | OE19 transfected with vector to express dCas9-KRAB under doxycycline control | |
| Cell line (H. sapiens) | CP-A-ERBB2 | This study | CP-A stably overexpressing ERBB2 | |
| Cell line (H. sapiens) | CP-A-empty | This study | CP-A cells containing an empty vector control | |
| Biological sample (H. sapiens) | Barrett’s oesophagus biopsies | Salford NHS FT | Freshly isolated from patients undergoing endoscopy | |
| Biological sample (H. sapiens) | Oesophageal adenocarcinoma biopsies | Salford NHS FT | Freshly isolated from patients undergoing endoscopy | |
| Transfected construct (human) | SmartPool siRNA against KLF5 | Horizon discovery | L-013571-00-0005 | |
| Transfected construct (human) | SmartPool siRNA against ERBB2 | Horizon discovery | L-003126-00-0005 | |
| Transfected construct (human) | SmartPool non-targeting siRNA | Horizon discovery | D-001810-10-0020 | |
| Transfected construct (human) | Full length non-targeting guide RNA | Synthego | 5’-GUAAGGCUAUGAAGAGAUAC-3’ | |
| Transfected construct (human) | Full length guide RNAs targeting KLF5 TSS | Synthego | 5’-GUGCGCUCGCGGUUCUCUCG-3’ 5’-AGGACGUUGGCGUUUACGUG-3’ 5’-GCGUCAAGUGUCAGUAGUCG-3’ |
|
| Antibody | Rabbit monoclonal KLF5 antibody | Abcam | ab137676 | (1:10000) for western blot; 5 ug for ChIP-seq |
| Antibody | Mouse monoclonal tubulin antibody | Sigma-Aldrich | T9026 | (1:2000) for western blot |
| Antibody | Spike-in antibody | Active Motif | 61686 | 1 ug for ChIP-seq |
| Antibody | Mouse monoclonal ErbB2 antibody | ThermoFisher | MA5-14057 | (1:1000) |
| Antibody | Mouse monoclonal AKT antibody | Cell signalling technology | 2920 |
(1:2000) |
| Antibody | Rabbit monoclonal phosphor-Akt (S473) antibody | Cell signalling technology | 4060S |
(1:2000) |
| Antibody | Rabbit monoclonal Erk1/2 antibody | Cell signalling technology | 4695S |
(1:1000) |
| Antibody | Mouse monoclonal phosphor-Erk1/2 (T202,Y204) | Cell signalling technology | 9106S |
(1:2000) |
| Antibody | Donkey anti-mouse secondary antibody (800CW) | Licor | 925 - 32212 |
(1:10,000) |
| Antibody | Donkey anti-rabbit secondary antibody (700CW) | Licor | 925–32213 | (1:10,000) |
| Recombinant DNA reagent | pX330-U6-Chimeric_BB-CBh-hSpCas9 (plasmid) |
Addgene | #42230 | AAVS guide RNA sequence 5’-GGGCCACTAGGGACAGGAT-3’ |
| Recombinant DNA reagent | pAAVS1-Puro-TRE-dCas9-KRAB-DNR (plasmid) | This study | pAS-4939 | |
| Recombinant DNA reagent | pHAGE-ERBB2 |
Addgene | 116734 |
|
| Recombinant DNA reagent | pHAGE-empty | This study | pAS-4940 | |
| Recombinant DNA reagent | pMD2.G | Addgene | 12259 | |
| Recombinant DNA reagent | psPAX2 | Addgene | 12260 | |
| Sequenced-based reagent | Primers | This study | See Supplementary file 11 | |
| Commercial assay or kit | Lipofectamine RNAiMAX |
Thermofisher | 13778150 | |
| Commercial assay or kit | Fugene HD | Promega | E2311 | |
| Commercial assay or kit | QuantiTect SYBR Green RT-PCR Kit | Qiagen | 204243 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | 74134 | |
| Commercial assay or kit | RNase-free DNase set | Qiagen | 79254 | |
| Commercial assay or kit | Ampure XP beads | Beckman Coulter Agencourt | A63881 | |
| Commercial assay or kit | TruSeq stranded RNA library kit v2 | Illumina | RS-122–2001 | |
| Commercial assay or kit | Nextera DNA library prep kit | Illumina | FC-121–1031 | |
| Commercial assay or kit | Nextera Index kit | Illumina | FC-121–1012 | |
| Commercial assay or kit | NEBNext high fidelity 2x PCR master mix | NEB | M0541 | |
| Commercial assay or kit | DNA Clean and Concentrator | Zymo | D4013 | |
| Commercial assay or kit | Polyfect | Qiagen | 301107 | |
| Commercial assay or kit | PEG-it | System Biosciences | LV810A-1 | |
| Commercial assay or kit | Polybrene | EMD Millipore | TR-1003 | |
| Chemical compound, drug | RS-1 | Sigma-Aldrich | R9782 | Used at final concentration 7.5 μM |
| Chemical compound, drug | SCR7 pyrazine | Sigma-Aldrich | SML1546 | Used at final concentration 1 μM |
| Chemical compound, drug | Doxycycline | Sigma-Aldrich | D3447 | Used at final concentration of 100 ng/mL |
| Chemical compound, drug | propidium iodide | Sigma | P4170 | Used at 50 μg/mL |
| Peptide, recombinant protein | RNase | Sigma | R4642 | Used at 100 μg/mL |
| Peptide, recombinant protein | EGF | ThermoFisher | 10450–013 | 5 μg/L |
| Peptide, recombinant protein | Bovine pituitary extract | ThermoFisher | 13028014 | Used at 50 mg/L |
| Software, algorithm | Trimmomatic | Bolger et al., 2014 | V0.34 | http://www.usadellab.org/cms/?page=trimmomatic |
| Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | v2.3.0 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml |
| Software, algorithm | Star | Dobin et al., 2013 | V2.5.4 | https://github.com/alexdobin/STAR |
| Software, algorithm | Macs2 | Zhang et al., 2008 | v2.1.1 | https://github.com/taoliu/MACS |
| Software, algorithm | Cufflinks | Tarapore et al., 2013 | v2.2.1 | http://cole-trapnell-lab.github.io/cufflinks/ |
| Software, algorithm | DEseq2 | Love et al., 2014 | V1.22.2 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html |
| Software, algorithm | TOBIAS | Bentsen et al., 2020 | v0.5.1 | https://github.com/loosolab/TOBIAS |
| Software, algorithm | featureCounts | Liao et al., 2014 | V1.6.2 | http://subread.sourceforge.net |
| Software, algorithm | FastQC | v0.11.4 | https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | |
| Software, algorithm | bedtools | Quinlan and Hall, 2010 | v2.26.0 | https://bedtools.readthedocs.io/en/latest/ |
| Software, algorithm | DeepTools | Ramírez et al., 2016 | V2.5.0 | https://deeptools.readthedocs.io/en/develop/ |
| Software, algorithm | GSEA | Subramanian et al., 2005 | V3.0 | http://software.broadinstitute.org/cancer/software/gsea/wiki/index.php/Main_Page |
| Software, algorithm | Homer | Heinz et al., 2010 | v4.9 | http://homer.ucsd.edu/homer/ |
| Software, algorithm | R | R Core Team (2018) | v3.5.1 | https://www.r-project.org/ |
| Software, algorithm | GraphPad Prism | V8.0 | www.graphpad.com | |
| Other | Crystal violet | Sigma Aldrich | HT90132 | Used at concentration of 0.1% |
| Other | Gibco RPMI 1640 | ThermoFisher | 52400 | |
| Other | Gibco fetal bovine serum | ThermoFisher | 10270 | |
| Other | Gibco penicillin/streptomycin | ThermoFisher | 15140122 | |
| Other | Keratinocyte SFM (1x) | ThermoFisher | 17005042 |