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| Status |
Public on Oct 03, 2022 |
| Title |
Single nuclei, Day1 MI, biol rep 4 |
| Sample type |
SRA |
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| Source name |
Cardiac
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| Organism |
Mus musculus |
| Characteristics |
tissue: Cardiac
genotype: WT
treatment: MI surgery
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| Extracted molecule |
total RNA |
| Extraction protocol |
Visium: Sections obtained from C57BL/6J (Jackson Laboratories) mice were imaged and processed for spatially resolved gene expression using the Visium Spatial Transcriptomics kit (10x Genomics). Samples were immediately snap-frozen in OCT using isopentane that was cooled in a liquid nitrogen bath. For cryosectioning, samples were equilibrated to −22 °C. 10 μm thick short axis sections were cut from the blocks onto Visium slides (10X Genomics) and processed according to the manufacturer’s protocol. Tissue permeabilization time was optimized at 30 minutes for infarcted mouse hearts. Hematoxylin and eosin (H&E) images generated during the Visium protocol were captured at 20x magnification on Nikon Eclipse Ti2-E widefield microscope and exported as tiled tiffs for analysis. Libraries were sequenced on the Illumina NovaSeq platform, and resulting data were processed using SpaceRanger (v.1.2.2, 10X Genomics)
Nuclei: Single nuclei were isolated from frozen tissue using a modified version of the protocol described (52). Mice hearts were weighed and minced before flash freezing with liquid nitrogen. Minced samples were resuspended in 0.5 ml nuclei lysis buffer (Millipore Sigma, Nuclei EZ prep, NUC101), 0.2 U/µl RNAse inhibitor (stock 40U/µl, Enzymatics Y9240L) and homogenized with a 2 ml dounce grinder for 10 strokes with A motor and at least 20 strokes with B motor (Sigma-Aldrich D8938). The lysates were resuspended with another 1 mL of nuclei lysis buffer and 2.5 minutes of incubation, and were filtered through 100 µm ,50 µm and 20 µm cell strainers (CellTrics filters 04-004-2318, 04-004-2317, 04-0042-2315). Then, they were centrifuged at 1000 x g for 5 min at 4°C to pellet nuclei. As Cui et al., 2020 described, the nuclear pellet was subsequently washed once in 6 mL of Sucrose buffer and centrifuged the cushioned suspension at 1,000 g for 10 min at 4°C to pellet nuclei. The pellet then was washed with 2ml of Nuclei Storage Buffer with 10 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) and centrifuged at 1000 x g for 5 min at 4°C (5mg/ml, Invitrogen D1306). 200 µl of nuclei wash buffer (freshly 2% bovine serum albumin (BSA) in 1xPBS, 0.2 U/µl RNAse inhibitor and 1mM of EDTA) were added and nuclei were resuspended. Fluorescence-activated cell sorting (FACS) were used to sort extracted nuclei. Nuclei was collected based on the separation between DAPIhigh and DAPIlow; After sorting using purity mode, DAPIhigh nuclei were pelleted at 1000 x g for 15 min at 4°C, resuspended in 2% BSA and trypan blue stained nuclei suspension were quality controlled and counted using hemocytometer (Hausser Scientific 3110V). Single-nucleus RNA-seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation, (10X Genomics). Paired end sequencing was performed on an Illumina Novaseq instrument. Low-level analysis, including demultiplexing, mapping to a reference transcriptome (mm10-1.2.0-premrna), and eliminating redundant UMIs, was performed with Cell Ranger 3.0.2 pipeline for 10X samples.
Single-nucleus RNA-seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation, (10X Genomics). Paired end sequencing was performed on an Illumina Novaseq instrument. Low-level analysis, including demultiplexing, mapping to a reference transcriptome (mm10-1.2.0-premrna), and eliminating redundant UMIs, was performed with Cell Ranger 3.0.2 pipeline for 10X samples.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
nuclear RNA
snd1_4
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the 10x Genomics Cell Ranger software v3.0.2 or Space Ranger v1.2.2
Further processing was done with Seurat v4.1(cell and gene filtering, hashtag identification, clustering, differential gene expression analysis).
Highly variable gene markers were identified using FindVariableFeatures from Seurat R package version 4.1
Assembly: GRCh38.p12.premrna2, mm10-1.2.0-premrna
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Oct 01, 2022 |
| Last update date |
May 16, 2023 |
| Contact name |
Kevin R King |
| Organization name |
UC San Diego
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| Department |
Medicine and Bioengineering
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| Lab |
King Lab
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| Street address |
9500 Gilman Dr. MC 0412
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE214611 |
Single Cell Spatial Transcriptomics Redefines the Borderzone induced by Myocardial Infarction and Mechanical Injury |
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| Relations |
| BioSample |
SAMN31119524 |
| SRA |
SRX17772394 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6613068_snd1_4.tar.gz |
44.4 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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