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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2024 |
| Title |
iwat, 60% n6 HFD, wt, rep 1 |
| Sample type |
SRA |
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| Source name |
inguinal white adipose tissue
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| Organism |
Mus musculus |
| Characteristics |
cell type: iWAT nuclei
tissue: inguinal white adipose tissue
diet: Research Diets #12492 60% HFD
genotype: WT
strain: C57Bl/6J
age: 18-22 weeks
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| Extracted molecule |
total RNA |
| Extraction protocol |
The nuclear isolation procedure was adapted from a from a previously published protocol. iWAT (200 mg/mouse) was placed in a petri-dish containing 500 μl of ice-cold nuclei preparation buffer (NPB: 10mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.05% IGEPAL CA-630 and 0.2 mM DTT (pH 8.0) in DPPC water) and minced with scissors for 4 min. The samples were then transferred to a 2ml glass Dounce homogenizer containing 500 μl NPB, followed by 10 strokes with a loose pestle (pestle A) and 10 strokes with a tight pestle (pestle B). The homogenates were filtered through a 70 μm cell strainer into 50 ml conical tube. The filters were rinsed with an additional 2 ml NPB, after which the homogenates were transferred to 5 ml Eppendorf tubes and centrifuged at 1,000 xg for 10 min at 4°C in swing-buckets. The supernatant and floating lipid layer were carefully aspirated, and the nuclear pellet was resuspended in 500 μL NPB containing 1 U/μl RNase inhibitor (New England Biolabs, M0314). Nuclear suspensions from 2 independent mice were combined at this point and centrifuged in 1.5 ml Eppendorf tubes at 1,000 xg for 10 min at 4°C. Finally, the nuclear pellet was collected in 150 μl resuspension buffer (2 mM MgCl2, 1% BSA, 1U/μl RNase inhibitor in DPBS. All solutions were filter-sterilized prior to use. Finally, the nuclei suspensions were filtered through a 40 μm cell strainer (Flowmi Cell Strainer, Belart, NJ). Following counting using a Bürker pattern counting chamber, 10,000 nuclei per genotype were submitted for sNuc-seq using the 10x Genomics system.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
The demultiplexing, barcoded processing, and gene counting were made using the Cell Ranger software v6.1.1
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Nov 28, 2022 |
| Last update date |
Dec 31, 2024 |
| Contact name |
Peter Tontonoz |
| E-mail(s) |
ptontonoz@mednet.ucla.edu
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| Phone |
(310) 206-4546
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| Organization name |
University of California, Los Angeles
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| Department |
Pathology & Laboratory Medicine
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| Lab |
Tontonoz Lab
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| Street address |
675 Charles E Young Dr.
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE218941 |
Transcriptional profiling of mature murine adipocytes of the inguinal white adipose tissue depot following LPCAT3 deletion and dietary PUFA manipulation [snRNA-seq] |
| GSE218943 |
Transcriptional profiling of mature murine adipocytes of the inguinal white adipose tissue depot following LPCAT3 deletion and dietary PUFA manipulation |
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| Relations |
| BioSample |
SAMN31898340 |
| SRA |
SRX18405975 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6760402_WT_n6_sWAT1.tar.gz |
61.5 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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