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| Status |
Public on Mar 31, 2020 |
| Title |
TFEB-wt Control-Rep_2 |
| Sample type |
RNA |
| |
|
| Source name |
BMDMs from LysM-Double-Cre x TFEB transgene- mice treated with DMEM only
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: LysM-Double-Cre x TFEB transgene- mice
age: 8 weeks
Sex: female
treatment: DMEM only
tissue: bone marrow-derived macrophage (BMDM)
|
| Treatment protocol |
Bone marrow was differentiated into macrophages using recombinant M-CSF. After one week of differentiation, macrophages were replated and treated with either DMEM only or DMEM supplemented with tumor-conditioned medium (serum-free DMEM supernatant exposed to EO771 tumor cells) and tumor lysate for 24 hours.
|
| Growth protocol |
Legs from eight week-old female LysM-Double-Cre x TFEB transgene+ and LysM-Double-Cre x TFEB transgene- mice were obtained from Babak Razani (Washington University in St. Louis).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated from bone marrow-derived macrophages using RNeasy Mini kit (Qiagen, Germantown, MD) following the manufacturer’s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with a NanoDrop 2000c Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >11.5 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray (G4852A-028005) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then immersed in acetonitrile and dried immediately by slowly removing the slide.
|
| Scan protocol |
Slides were scanned immediately after washing on an Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green at 20 bit).
|
| Description |
Gene expression of bone marrow-derived macrophages from LysM-Double-Cre x TFEB transgene- mice treated with DMEM only
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid 028005_D_F_20130207) to obtain background subtracted and spatially detrended Processed Signal intensities. Data was uploaded into Genespring version 14.8 where it was log2 transformed, quantile normalized and base line transformed using the median of all samples. Data was filted by flags in a way that at least 75% of the samples in any of the experimental groups had DETECTED flags. Then, differentially expressed genes were determined.
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| |
|
| Submission date |
Oct 29, 2019 |
| Last update date |
Apr 01, 2020 |
| Contact name |
Diego Altomare |
| Organization name |
University of South Carolina
|
| Department |
Department of Drug Discovery and Biomedical Sciences
|
| Lab |
Functional Genomics Core
|
| Street address |
715 Sumter Street, Room 617
|
| City |
Columbia |
| State/province |
SC |
| ZIP/Postal code |
29208 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE139554 |
Bone marrow LysM-Cre mediated TFEB overexpression significantly affected the response of bone marrow-derived macrophages to tumor material, decreasing both the expression of typical M2-like macrophage associated genes and inflammatory response genes. |
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