|
| Status |
Public on Apr 17, 2023 |
| Title |
SUM159_pLV-KRAB+all-4-gRNAs_rep3 [All.gRNA3] |
| Sample type |
genomic |
| |
|
| Source name |
SUM159 cells, transfected with pLV-KRAB dCas9/CRISPRi system + 4 targeting gRNAs against the ZEB1 promoter
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SUM159
gender: Female
cell type: triple negative breast cancer cell line
|
| Treatment protocol |
Lentiviral particles were produced using HEK293T with co-transfection using third generation VSVG (Addgene plasmid #12259), GAG/POL (Addgene plasmid #12260) and pLV dCas9-KRAB +/-gRNA as previously described [doi:10.1039/c9sc01432b & 10.1016/j.omtn.2018.12.003]. Four days following transduction, 1.25 µg/mL puromycin was used to select SUM159 cells.
|
| Growth protocol |
F12 Medium supplemented with 0.6% 1M Hepes, 5 µg/mL insulin, 1 µg/mL hydrocortisone, 1% Anti-Anti and 10% heat inactivated FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed twice in PBS before being collected. DNA was extracted with the Monarch Genomic DNA Purification kit (New England Biolabs). After QC, 500ng of purified DNA was processed using Illumina Methylation EPIC 850k Array.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
Standard Illumina Protocol
|
| |
|
| Hybridization protocol |
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol
|
| Scan protocol |
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
|
| Description |
SUM159 (triple negative breast cancer) cell line transfected with a pLV-KRAB dCas9/CRISPRi system + 4 gRNAs targeting the ZEB1 promoter
|
| Data processing |
Methylation data analysis were conducted using the minfi R package to read.idat files into R (version 3.6.3), and the missMethyl R package was used for data processing and normalization. Specifically, we identified low quality probes and removed them from the data normalized by SWAN. We then extracted β and M-values (with an offset of 100 for calculating M values). Data quality control was performed using PCA and MDS plots as well as histograms of β values, probe-level differential methylation analysis was performed on M-values using limma, and probes with an adjusted p-value < 0.05 and absolute log fold change; 0.5 were considered as differentially methylated probes (DMPs). To map probes to their associated genes and gene regions, we used the IlluminaHumanMethylationEPICanno.ilm10b2.hg19 R package.
|
| |
|
| Submission date |
Aug 01, 2022 |
| Last update date |
Apr 18, 2023 |
| Contact name |
Joe Cursons |
| Organization name |
Monash University
|
| Department |
Biomedicine Discovery Institute
|
| Street address |
19 Innovation Walk
|
| City |
Clayton |
| State/province |
VIC |
| ZIP/Postal code |
3800 |
| Country |
Australia |
| |
|
| Platform ID |
GPL23976 |
| Series (2) |
| GSE210273 |
Synthetic epigenetic reprogramming of mesenchymal to epithelial states using the CRISPR/dCas9 platform in triple negative breast cancer [methylation array] |
| GSE210277 |
Synthetic epigenetic reprogramming of mesenchymal to epithelial states using the CRISPR/dCas9 platform in triple negative breast cancer |
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