|
| Status |
Public on Nov 22, 2021 |
| Title |
Dnmt1c/chip MDP rep2 |
| Sample type |
RNA |
| |
|
| Source name |
MDPs flow-sorted from the bone marrow of Dnmt1c/chip mice
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Dnmt1c/chip
cell type: MDPs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
BM cells were obtained from adult mice by thoroughly flushing femurs and tibiae with PBS (PAA) supplemented with 1 % FSC (Biochrom). Single cell suspensions were generated by mashing the organ through a cell strainer of 40 µm diameter. Suspension of cells were incubated for 5 min with ice-cold ACK buffer (0.15 M NH4Cl, 10 M KHCO3, 0.1 mM EDTA, pH 7.3) to remove erythrocytes. Cells were first incubated for 10 min in PBS supplemented with 1% FCS and purified CD16/32 antibody to block Fc binding. Antibody labeling was conducted for 10 min on ice in the dark. Discrimination of dead cells was performed by propidium iodide (PI, 0.1 mg/ml, Sigma Aldrich) shortly before measurement. MDPs (Lin-cKithiCD115+CD11b-Ly-6C-) and cMoPs (Lin-cKithiCD115+CD11b-Ly-6C+) were flow-sorted from 8-12 weeks old Dnmt1+/+ and Dnmt1c/chip mice with an FACS Aria III (BD) cytometer equipped with FACSDiva software. The RNAeasy Kit (Qiagen) was used for extraction of RNA according to the supplier’s instructions. DNAse I digestion was performed as part of the protocol to remove residual DNA from the RNA preparation. RNA was stored at -80 °C.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix GeneChip 3’ IVT Express Protocol was used for the preparation of biotinylated amplified RNA (aRNA) from 300ng of total RNA.
|
| |
|
| Hybridization protocol |
6 µg of fragmented and labeled aRNA were hybridized to Affymetrix Mouse Genome 430 PM peg plate arrays. For hybridization, washing and staining an Affymetrix GeneTitan system was used.
|
| Scan protocol |
GeneChip arrays were scanned using an Affymetrix GeneTitan system controlled by the Affymetrix GeneChip Command Console software (AGCC).
|
| Data processing |
Data was analyzed with the Bioconductor package version v.3.5.1. The CEL files containing the raw expression intensity values for each probe on the array were opened using the affy package and normalized using the function called Robust Multi-Array Average (RMA). The linear models for microarray data (LIMMA) package was used to find differentially expressed genes. A cutoff of more than 0.58 logFC and p-value of less than 0.05 was used to call genes as differentially expressed between two conditions.
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| |
|
| Submission date |
Dec 16, 2020 |
| Last update date |
Nov 22, 2021 |
| Contact name |
Sweta Talyan |
| E-mail(s) |
[email protected]
|
| Organization name |
MPI
|
| Street address |
Ludwigstraße 43
|
| City |
Bad Nauheim |
| ZIP/Postal code |
61231 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17400 |
| Series (1) |
| GSE163295 |
Whole transcriptome profiling of macrophage-DC progenitors (MDPs) and common monocyte progenitors (cMoPs) from Dnmt1+/+ and Dnmt1c/chip mice |
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