|
| Status |
Public on Apr 24, 2019 |
| Title |
M2_BMDM_scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
bone marrow derived macrophages
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/j
tissue: bone marrow
age: 17~18 weeks
|
| Treatment protocol |
refer to the description column of the SAMPLES section
|
| Growth protocol |
Male mice (C57BL6/j) were housed in a 12/12-hour light: dark cycle and provided ad libitum access to food and water for the duration of the study unless stated otherwise. Male mice of 5-6 weeks of age were used for feeding analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
refer to the description column of the SAMPLES section
Single-cell gel beads in emulsion (GEMs) macrophages were generated on a GemCode Single Cell Instrument (10x Genomics). Single-cell RNA-seq cDNA libraries were prepared using Chromium™ Single Cell 3' Reagent Kit v2 (10x Genomics) according to manufacturer's protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
For dietary feeding studies, mice were fed with a standard chow diet (18% fat calories, 24% protein calories, and 58% of carbohydrate calories; Cat No. 2918, Envigo. Teklad.) for 12 weeks. bone marrow cells were collected from femur and tibia bone followed by erythrocyte lysis with ammonium chloride solution (8.3 g/L ammonium chloride, 1.0 g/L potassium bicarbonate, and 0.009% EDTA) and seeded in tissue culture plates at a concentration of 0.15 x 106/cm2. Cells were induced for differentiation to monocytes in BMDM growth medium (Iscove's Modified Dulbecco's Medium (IMDM) + 10% FBS + 15% L-929 cell (ATCC, CCL-1)) culture supernatant for 7 days; fresh BMDM growth medium was replaced on day 3. Maturation and purity of BMDMs were evaluated on day 7 using flow cytometry analysis with fluorescence conjugated antibodies against CD11b and F4/80. To induce polarization, BMDMs were stimulated 20 ng/mL IL4 and 20 ng/mL IL13 and collected as M2 activation state.
|
| Data processing |
The amplified cDNA from each channel of the Chromium System was used to construct an Illumina sequencing library and sequenced on a HiSeq 4000 with 150 cycle sequencing (asymmetric reads per 10x Genomics).
Illumina basecall files (*.bcl) were converted to FASTQs using CellRanger v1.3 (10X Genomics), which uses bcl2fastq v2.17.1.14.
FASTQ files were then aligned to mm10 mouse reference genome and transcriptome using the CellRanger v1.3 software pipeline with default parameters , which demultiplexes the samples and generates a gene versus cell expression matrix based on the barcodes and assigned unique molecular identifiers (UMIs) that enable determining from which individual cell RNA molecule originated.
Genome_build: mm10
Supplementary_files_format_and_content: Market Exchange Format (MEX) matrices (.mtx) containing the UMI counts for each cell; TSV files (.tsv) with genes and barcode sequences corresponding to row and column indices of the MEX file, respectively
|
| |
|
| Submission date |
Jul 16, 2018 |
| Last update date |
Apr 25, 2019 |
| Contact name |
Beiyan Zhou |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Connecticut
|
| Department |
Immunology
|
| Street address |
263 Farmington Ave.
|
| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE117176 |
Single cell RNA sequencing of murine bone marrow derived macrophages and adipose tissue macrophages from lean and obese mice |
|
| Relations |
| BioSample |
SAMN09665938 |
| SRA |
SRX4394828 |
