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| Status |
Public on Oct 29, 2024 |
| Title |
BTBR_ob_section2 |
| Sample type |
SRA |
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| Source name |
Kidney
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| Organism |
Mus musculus |
| Characteristics |
tissue: Kidney
region: Sagittal
model: BTBR ob/ob
age (weeks): 10
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| Extracted molecule |
total RNA |
| Extraction protocol |
As each region was sampled, the tissue was chopped into 1mm x1mm cubes and placed in 0.25mg/ml liberase TH (Roche Diagnostics, Indianapolis, USA) dissociation medium. Following further dissection, the samples were incubated at 37°C for 1 hour at 600rpm. Samples were regularly triturated during the incubation period using a 1ml pipette, after which 10% heat- inactivated FBS RPMI was added to stop the digestion. Centrifugation at 500 g for 5 minutes at room temperature with the removal of the supernatant, was followed by the addition of ACK lysing buffer to remove erythrocytes (Thermo Fisher Scientific, Waltham, USA). Following centrifugation, the resulting cell pellet was incubated with Accumax at 37°C for 3 minutes (Innovative Cell Technologies Inc, San Diego, USA). 10% FBS RPMI was again used to neutralize the Accumax and centrifugation was followed by resuspension of the cell pellet in 0.4% BSA/PBS. The single cell suspension was filtered using a 30um CellTrics filter (Sysmex America Inc, Lincolnshire, USA) with cell viability and concentration determined using trypan blue and the Auto T4 cellometer (Nexcelom Bioscience LLC, Lawrence, USA).
17,500 cells were loaded into the 10x Genomics microfluidic system/onto the 10x Genomics platform to achieve a targeted recovery of 10,000 cells (10x Genomics, Pleasanton, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
10x Genomics
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
cellranger-6.1.2, CellBender remove-background 0.2.0, scDblFinder_1.6.0
Samples raw base calls demultiplexing, barcoded processing, sequence alignment and gene counting were made using the Cell Ranger software 6.1.2 function. Raw sequence reads were aligned to the latest mouse reference genome (mm10).
Counts were pre-processed for ambient RNA and doublet removal using CellBender remove-background 0.2.0
Doublet removal was performed using scDblFinder_1.6.0 in the R programing language
Downstream analysis were performed in R.
Assembly: mm10_v3.0.0
Supplementary files format and content: h5 files from CellRanger and CellBender, respectively.
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| Submission date |
Jun 07, 2022 |
| Last update date |
Oct 29, 2024 |
| Contact name |
Ayshwarya Subramanian |
| E-mail(s) |
[email protected]
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| Organization name |
Broad Institute
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| Department |
Klarman Cell Observatory
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| Street address |
415 Main Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE205592 |
Obesity-instructed TREM2-high macrophages identified by comparative analysis of diabetic mouse and human kidney at single-cell resolution [scRNA-seq: DKD_mouse] |
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| Relations |
| BioSample |
SAMN28900133 |
| SRA |
SRX15624410 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6215282_BTBR_ob_section2_out.h5 |
129.2 Mb |
(ftp)(http) |
H5 |
| GSM6215282_BTBR_ob_section2_raw_feature_bc_matrix.h5 |
17.8 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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