 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 07, 2023 |
| Title |
10x snRNA-seq on the mouse liver (7b2a3b) |
| Sample type |
SRA |
| |
|
| Source name |
Liver (P56)
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Liver (P56)
genotype: CD1
sample state: Fresh
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The liver nuclei were isolated following the protocol described by Thrupp et al., 2020. For the fresh samples, 200 mg of fresh mouse liver tissue was minced and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (320 mM Sucrose, 5 mM CaCl2, 3 mM Magnesium Acetate, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1% Igepal, 0.1 mM PMSF, 1 mM βME and 0.2 U/µL RNasin Plus RNase Inhibitor (Promega). For the frozen samples, a piece of 200 mg of liver tissue was sectioned on dry ice and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer and let to thaw for 5 minutes. From this step onward, both the fresh and frozen tissue were homogenized with 10 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). Next, we added 1.65 mL of homogenization buffer to top the homogenate up and mixed it with 2.65 mL of gradient medium (5 mM CaCl2, 50% Optiprep, 3 mM Magnesium Acetate, 10 mM Tris, 0.1mM PMSF, 1mM βME). 4 mL of 29% Iodoxanol cushion was prepared with a diluent medium (250 mM sucrose, 150 mM Potassium chloride, 30 mM Magnesium chloride, 60 mM Tris pH 8) and added into an ultracentrifuge tube. Following, 5.3 mL of sample (mixed with the homogenization buffer and the gradient medium) was gently layered on top of the 29% Iodoxanol cushion. Samples were centrifuged in a SW41Ti rotor at 7700 x g, 4°C for 30 min and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 200 μL of resuspension buffer (1x PBS, 1% BSA and 0.2 U/μL Rnasin Plus RNase Inhibitor (Promega)) and transferred to a 1.5 mL Eppendorf tube. 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology, and presence of clumps/debris.
Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3’ Reagent v3 Kits (10x Genomics) according to the manufacturer’s protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Mouse_1_Fresh
Single-cell RNA-seq (10X Genomics)
NovaSeq6000/HiSeq4000
|
| Data processing |
The generated fastq files were processed with cellranger (v1.0.0) count function. Reads were aligned to “pre-mRNA” Mus musculus reference genome, that included both exons and introns (10x Genomics, see https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references#premrna).
10x snRNA-seq and 10x multiome (gene expression) runs were analyzed first independently using VSN-pipelines (v0.27.0). Briefly, cells with at least 350 genes expressed and a percentage of mitochondrial reads below 10% were kept. Scanpy (v1.8.2) was run with default parameters, using the number of principal components automatically selected by VSN-Pipelines and using Leiden clustering with resolutions 0.4, 0.6 and 0.8. Hepatocyte clusters with low gene expression and high percentage of mitochondrial reads were removed, as well as doublets called with Scrublet (v0.2.3) [REF]. The samples were merged, obtaining 29,798 high-quality cells, and reanalyzed with VSN-Pipelines.
Assembly: mm10
Supplementary files format and content: scrna_counts.tsv: Tab-separated flat text file containing genes as rows, cells as columns and counts for each gene in each cell as values.
|
| |
|
| Submission date |
Nov 21, 2022 |
| Last update date |
Sep 07, 2023 |
| Contact name |
Carmen Bravo Gonzalez-Blas |
| Organization name |
VIB-KU Leuven Center for Brain & Disease Research
|
| Street address |
Herestraat 49
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE218465 |
Enhancer grammar of liver cell types and hepatocyte zonation states [scRNA-seq] |
| GSE218472 |
Enhancer grammar of liver cell types and hepatocyte zonation states |
|
| Relations |
| BioSample |
SAMN31819793 |
| SRA |
SRX18332041 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
