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| Status |
Public on Jun 27, 2019 |
| Title |
AB2577 |
| Sample type |
SRA |
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| Source name |
White adipose tissue
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| Organism |
Mus musculus |
| Characteristics |
strain: Db/Db C57BL/6
treatment (diet): Normal chow
mouse age (weeks): 7
time point (weeks): 7
tissue subtype: epididymal (visceral) adipose tissue (EAT)
cell type: total immune cells
selection marker: CD45+
replicate id: 9
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| Treatment protocol |
[treatment protocol (1)] At age 8-9 weeks, for some mice the normal chow was replaced with a high fat diet (HFD; irradiated Rodent Diet With 60 kcal% Fat, D12492i Research Diets Inc., New Brunswick, NJ).
[treatment protocol (2)] Founding Trem2-/- knock-out (KO) breeder mice were crossed with wild-type (WT) mice at the Weizmann Institute animal facility to produce second-generation cohorts of WT and KO littermates. F1 offspring was bred to produce homozygous WT or KO, some of which were also set to feed on HFD. Heterozygous F2 mice were not used for experiments.
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| Growth protocol |
8-week old mice, housed at the Weizmann Institute animal facility.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were euthanized and nd perfused immediately through the left ventricle of the heart with 20 ml of phosphate-buffered saline (PBS) to remove circulating leukocytes from the tissue. The epididymal (visceral) adipose tissue (EAT) was readily located and excised right above the epididymis. The subcutanous adipose tissue (SAT) was obtained from the inguinal fat depot. The tissue was placed in a 50 ml tube with 10 ml DMEM without phenol red at room temperature, cut into tiny bits with scissors, incubated with collagenase II at 37°C for 20 min. while gently agitating, filtered through a 100-μm cell strainer, and spun down at 500g for 10 min with low acceleration/brake, beginning at room temperature and cooling to 4°C. Cells were resuspended in 500 μl RBC lysis solution (Sigma) and incubated on ice for 2-5 min. (depending on the initial amount of tissue) and washed. The resulting cell suspension was incubated with anti-CD45 or a cocktail of antibodies for selected markers.
Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. Briefly, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014};
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Note: Second read contained only cell and molecule barcodes. This information was appended to the fastq entry header
bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Pool-barcode and well-barcode-RMT were extracted from the first and second end of the read (respectively) and concatenated to the fastq header, delimited by a underscore i.e. POOL_BARCODE_WELL_BARCODE_RMT while "NNNNNN" was used as a place holders if plate barcode was not used.
Reads were separated by POOL_BARCODE_WELL_BARCODE header data, allowing 1 sequencing error. This process created a single fastq file for each source well.
Genome_build: mm10
Supplementary_files_format_and_content: [AB*.txt] tab-delimited text files include mRNA molecule count values for each Sample
Supplementary_files_format_and_content: [metadata.txt] file associating each single cell with its amplification batch and index sorting readouts
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| Submission date |
Mar 19, 2019 |
| Last update date |
Jun 27, 2019 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
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| Phone |
972-8-9343338
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| Organization name |
Weizmann Institute of Science
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| Department |
Immunology
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| Street address |
234 Herzl st.
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| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE128518 |
Lipid-associated macrophages control metabolic homeostasis in a Trem2-dependent manner |
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| Relations |
| BioSample |
SAMN11165390 |
| SRA |
SRX5541254 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3678359_AB2577.txt.gz |
768.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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