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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jun 13, 2016 |
Title |
CSF1R-3_L4.N709 |
Sample type |
SRA |
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Source name |
Dicer_x_LysM.Cre (D–/–)
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Organism |
Mus musculus |
Characteristics |
genotype/variation: myeloid-cell-specific Dicer1 gene deletion protocol: inoculated Lewis lung carcinoma (LLC) cells subcutaneously in mice cell type: tumor-associated macrophages (F4/80+ cells)
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Treatment protocol |
To explore the role of DICER in the development, activation and immunological functions of TAMs, we crossed homozygous LysM-Cre (Clausen et al., 1999) with Dicerlox/lox (Harfe et al., 2005) mice to obtain mice with myeloid-cell-specific Dicer1 gene deletion (LysM-Cre;Dicer–/–, referred to as D–/–). These mice were then backcrossed to LysM-Cre to obtain the control LysM-Cre mice; Dicer+/+ mice (referred to as D+/+). Both LysM-Cre and Dicerlox/lox mutations were always homozygous in our experiment. We then inoculated Lewis lung carcinoma (LLC) cells subcutaneously (s.c.) in D–/– and control D+/+ mice. Once the tumors were established, we isolated by fluorescence-activated cell sorting (FACS) tumor-associated macrophages (F4/80+ cells).
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were homogenized in 700 µl Qiazol reagent (Qiagen). 140 ul of chloroform was added to the homogenate and the mixture centrifuged at 16,000 x g for 5 min at 4°C in Phase Lock Tubes (Qiagen). RNA was subsequently extracted from the aqueous phase using the RNeasy Micro kit (Qiagen) according to manufacturer’s instructions. Genomic DNA was removed using the RNase free DNase set (Qiagen) during the extraction. RNA was quality controlled on Agilent RNA 6000 pico chips (Agilent Technologies). High quality RNA (RIN >7) was obtained for all samples. SMARTer ultra low RNA kit for Illumina sequencing (Clontech) was used to prepare and amplify cDNA from two ng of total RNA according to manufacturer’s instructions. One ng of amplified cDNA was then subjected to Nextera XT library preparation (Illumina) according to manufacturer’s instructions. Sequencing libraries were quantified using the Kapa Library Quantification kit (Kapa Biosystems) and quality controlled by capillary electrophoresis on a Bioanalyzer using High Sensitivity chips (Agilent Technologies). Libraries were sequenced on a HiSeq2500 sequencer (Illumina) for 2 x 50 cycles using version 4 cluster generation kits and version 4 sequencing reagents (Illumina).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Raw read data was aligned against mouse transcriptome (MouseEnsembl release 61) using Bowtie2 (Langmead and Salzberg, 2012). Splice junctions were defined as available in Ensembl v75. Read mappings were then mapped to genes and transcripts based on the transcript models annotated in Ensembl v75 using in-house tools and, again using in-house software, RPKM values were computed as described previously (Mortazavi et al., 2008). Genome_build: MouseEnsembl release 61 Supplementary_files_format_and_content: tab-delimited text file includes RPKM values for each sample
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Submission date |
Dec 28, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Chia-Huey Ooi |
Organization name |
F. Hoffmann-La Roche Ltd.
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Street address |
Grenzacherstrasse 124
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City |
Basel |
ZIP/Postal code |
4070 |
Country |
Switzerland |
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Platform ID |
GPL17021 |
Series (1) |
GSE76356 |
DICER controls macrophage polarization and tumor response to immunotherapy |
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Relations |
BioSample |
SAMN04372250 |
SRA |
SRX1503008 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1982044_CSF1R-3_L4.N709.expression.txt.gz |
747.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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