|
| Status |
Public on Jul 28, 2024 |
| Title |
genomic DNA from A549_EV |
| Sample type |
genomic |
| |
|
| Source name |
A549, empty vector
|
| Organism |
Homo sapiens |
| Characteristics |
tumor type: lung carcinoma
genotype: empty vector
|
| Treatment protocol |
A549 and MDA-MB-231 cells with lentivirus-mediated stable overexpression of STELLA orthologs were harvested on Day 9 post virus-transduction. Mice with HCT116 xenograft tumors were peritumorally injected every other day by the indicated LNP formulations and the tumor tissues were isolated on Day 18.
|
| Growth protocol |
A549 and MDA-MB-231 cells were maintained in DMEM complete medium. The tumor tissues were isolated from the mice bearing HCT116 xenograft tumors.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted and purified from cell pellets using the Promega Wizard Genomic DNA Purification Kit according to the manufacturer's instructions. Genomic DNA quality was assessed by low concentration agarose gel (0.6%) electrophoresis and spectrometry of OD260/280 and OD 260/230 ratio.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
DNA bisulfite conversion was carried out using EZ DNA Methylation Kit (Zymo Research) by following manufacturer's manual with modifications for Illumina Infinium Methylation Assay. Briefly, 0.5~1.0 ug of genomic DNA was first mixed with 5 ul of M-Dilution Buffer and incubate at 37C for 15 minutes and then mixed with 100 ul of CT Conversion Reagent prepared as instructed in the kit's manual. Mixtures were incubated in a thermocycler with 16 thermal cycles at 95C for 30 seconds and 50C for one hour. Bisulfite-converted DNA samples were loaded onto 96-column plates provided in the kit for desulphonation and purification. Concentration of eluted DNA was measured using Nanodrop-1000 spectrometer. Bisulfite-converted DNA was analyzed using Illumina Infinium MethylationEPIC v2.0 BeadChip Kit (20087708) by following manufacturer's manual. Beadchip contains more than 935,000 CpG loci in human genome. Briefly, 4 ul of bisulfite-converted DNA was added to a 0.8 ml 96-well storage plate (Thermo Scientific), denatured in 0.014N sodium hydroxide, neutralized and amplified with kit-provided reagents and buffer at 37C for 20-24 hours. Samples were fragmented using kit-provided reagents and buffer at 37C for one hour and precipitated by adding 2-propanol. Re-suspended samples were denatured in a 96-well plate heat block at 95C for 20 minutes. 12 ul of each sample was loaded onto a chip and the chips were assembled into hybridization chamber as instructed in the manual. After incubation at 48C for 16-20 hours, chips were briefly washed and then assembled and placed in a fluid flow-through station for primer-extension and staining procedures.
|
| |
|
| Hybridization protocol |
Bisulfite-converted DNA was analyzed using Illumina Infinium MethylationEPIC v2.0 BeadChip Kit by following manufacturer's manual. Beadchip contains more than 935,000 CpG loci in human genome. Briefly, 4 ul of bisulfite-converted DNA was added to a 0.8 ml 96-well storage plate (Thermo Scientific), denatured in 0.014N sodium hydroxide, neutralized and amplified with kit-provided reagents and buffer at 37C for 20-24 hours. Samples were fragmented using kit-provided reagents and buffer at 37C for one hour and precipitated by adding 2-propanol. Re-suspended samples were denatured in a 96-well plate heat block at 95C for 20 minutes. 12 ul of each sample was loaded onto a chip and the chips were assembled into hybridization chamber as instructed in the manual. After incubation at 48C for 16-20 hours, chips were briefly washed and then assembled and placed in a fluid flow-through station for primer-extension and staining procedures.
|
| Scan protocol |
Polymer-coated chips were image-processed in Illumina iScan scanner.
|
| Description |
A549 cells stably expressing empty vector
|
| Data processing |
Data were extracted using Illumina GenomeStudio V2011.1 software. Methylation status of each CpG site was presented as beta value based on following definition: beta value = (signal intensity of methylation-detection probe)/(signal intensity of methylation- detection probe + signal intensity of non-methylation-detection probe + 100).
Matrix_processed.txt: Average Beta
Matrix_signal_intensities.txt: Unmethylated and methylated signal intensities
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| |
|
| Submission date |
Jul 26, 2024 |
| Last update date |
Jul 28, 2024 |
| Contact name |
Wenbin Gu |
| E-mail(s) |
epoch1991@gmail.com
|
| Organization name |
Guangzhou Institutes of Biomedicine and Health
|
| Department |
Chinese Academy of Sciences
|
| Lab |
Center for Chemical Biology and Drug Discovery
|
| Street address |
190 Kaiyuan Avenue, Huangpu District, Guangzhou
|
| City |
Guangzhou |
| ZIP/Postal code |
510530 |
| Country |
China |
| |
|
| Platform ID |
GPL33022 |
| Series (1) |
| GSE273176 |
DNA Methylome Analysis of Human Cancer Cells with STELLA Protein Overexpression and the Xenograft Tumor Tissues from the Mice Treated by the Indicated LNP Formulations |
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