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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 12, 2024 |
| Title |
Sp140 KO Bystander and naïve cells, mRNA-derived cDNA [RVDK002C] |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
cell type: myeloid cells
treatment: Mtb infected and naïve
genotype: Sp140 KO
library type: mRNA
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
FACS isolated cells were pooled and loaded into 3 different lanes on a Chromium Next GEM Chip. Lane 1 was loaded with Mtb-infected cells from all 3 B6 and 3 Sp140–/– lungs. Lane 2 was loaded with bystander myeloid cells from the 3 infected B6 lungs as well as the myeloid cell mixture from the 2 naïve B6 lungs. Lane 3 was loaded with bystander myeloid cells from the 3 infected Sp140–/– lungs as well as the myeloid cell mixture from the 2 naïve Sp140–/– lungs. All 3 lanes of the Chromium Next GEM Chip were super-loaded with 29000 cells with a target of 14800 single cells per lane,
The scRNA-sequencing libraries were generated using the v3.1 chemistry Chromium Single Cell 3’ Reagent Kit (10X Genomics) largely following this kits protocol and the CITE-seq protocol. 0.5 U/µL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) was added to single cell RT master mix during the loading step and 1 µL of ADT and HTO additive primers (0.2 µM stock) were added during the cDNA amplification. Following cDNA, ADT, and HTO purification, samples were decontaminated by 2 rounds of centrifugation through 0.2 µM filter microcentrifuge tubes and then removed from the BSL3. Library preparations were completed outside of the BSL3 following the 10X Genomics protocol for the cDNA and the CITE-seq and Cell Hashing Protocol for the ADT and HTO libraries. Quality control of the libraries was performed with a Fragment Analyzer (Agilent). The mRNA, ADT, and HTO libraries were pooled at the following proportions: 85% mRNA, 9% ADT, and 6% HTO. Libraries were sequenced on a NovaSeq 6000 (Illumina) using two lanes of a S1 flow cell and the following cycles read 1 (28 cycles), i7 index (10 cycles), i5 index (10 cycles), read 2 (90 cycles).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
polyA RNA
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| Data processing |
library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)
Raw sequencing reads for the mRNA libraries were processed into raw count matrices with CellRanger version 4.0.0 (10X Genomics). The ADT and HTO libraries were processed into raw count matrices with CITE-Seq-Count version 1.4.3.
The raw counts for mRNA, ADT, and HTO were analyzed using Seurat v4.1.1 using default settings for normalizing the data, finding variable features, and scaling the data. HTO demultiplexing was performed with the HTODemux function. Data was filtered to only include single cells with between 200 and 4500 genes and less than 5% mitochondrial reads. The resulting datasets were integrated together using 30 dimensions for the FindIntegrationAnchors function and 30 dimensions for the IntegrateData function. The data was then scaled and analyzed by PCA with 30 principal components followed by UMAP analysis with 30 dimensions. Clustering was performed by using 30 dimensions with the FindNeighbors function and a resolution of 0.8 for the FindClusters function.
To improve resolution for clustering innate immune cells, weighted nearest neighbor analysis was used to combine the protein data (ADTs) and the mRNA data when clustering cells. For this analysis, variable ADT features were identified and then normalized using centered log ratio transformation and a margin of 2. The normalized ADT data was then scaled and analyzed by PCA. The ADT and mRNA data was then combined with the FindMultiModalNeighbors function using 30 dimensions for the mRNA and 10 for the protein. The resulting dataset was analyzed by UMAP and clusters were identified with the FindClusters function using algorithm 3 and a resolution of 1.5.
Assembly: mm10
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
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| Submission date |
Oct 18, 2022 |
| Last update date |
Jan 12, 2024 |
| Contact name |
Dmitri I Kotov |
| E-mail(s) |
dkotov@berkeley.edu
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| Organization name |
University of California, Berkeley
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| Department |
Molecular and Cell Biology
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| Lab |
Russell Vance
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| Street address |
415 Weill Hall
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE216023 |
CITE-seq analysis of Mycobacterium tuberculosis infected B6 and Sp140-deficient mouse lungs |
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| Relations |
| BioSample |
SAMN31351623 |
| SRA |
SRX17937616 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6656184_RVDK002C_barcodes.tsv.gz |
81.1 Kb |
(ftp)(http) |
TSV |
| GSM6656184_RVDK002C_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM6656184_RVDK002C_matrix.mtx.gz |
93.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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