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| Status |
Public on Jul 01, 2024 |
| Title |
Whole Blood, Lane 1, scRNAseq |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell type: Mix
treatment: 24c5 IgG1, 24c5 SEHFST LS, No Ab, and Uninfected
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| Treatment protocol |
Blood was infected with Mtb and treated with antibodies.
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| Growth protocol |
Whole-blood from healthy 3 human donors was collected the day of the experiment in acid citrate dextrose anti-coagulant tubes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Blood from each donor was added at a 1:9 ratio to ACK lysis buffer (Quality Biological, 10128-802) and incubated for 10min at RT. Cells were centrifuged at 400g for 5min, and the supernatant was discarded. 10mL of ACK lysis buffer was added to the cell pellet to repeat the lysis procedure. Cells were centrifuged at 400g for 5min, and the supernatant was discarded. Samples were washed twice with PBS buffer and counted before MULTI-seq barcoding
In brief, samples were barcoded with 2.5μM of the LMO anchor and barcode for 5min on ice in PBS before adding 2.5μM of the LMO co-anchor and incubating for an additional 5min. Samples were quenched with 1% BSA in PBS and washed once. Samples were pooled and 0.5U/μL RNase inhibitor (Roche) was added. Pooled samples were then loaded into two lanes using the 10X Genomics NextGEM Single Cell 3’ kit v3.1 per the manufacturer’s protocol. cDNA was inactivated at 95°C for 15min prior to biosafety level 3 removal for library construction. Libraries were sequenced on a NextSeq500 (Illumina). FASTQ files were processed using CellRanger v6.1.2 to generate gene expression count matrices and deMULTIplex to generate LMO barcode count matrices.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
scRNA - 10x genomics
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| Data processing |
LMO barcode and gene expression count matrices were analyzed using R (v4.0.3) and Seurat (v4.0.0). Cells were identified using emptyDrops (DropletUtils) were demuxed using HTODemux (Seurat) and hashedDrops (DropletUtils). Each lane was subject to demultiplexing and quality control separately then merged for downstream analyses. Cells with less than 300 unique genes detected were excluded. Additional cells were excluded based on the assessment of cluster-specific technical metrics (percent of mitochondrial reads per cell and number of UMIs per cell). Counts were normalized using the default parameters from NormalizeData (Seurat), i.e. scaling by 10,000 and log normalization. 3,000 variable features were used for PCA. SLM clustering was performed using FindClusters (Seurat) on the shared nearest neighbor graph generated from FindNeighbors (Seurat) using 30 principal components and k=20. Cell type annotation was based on expert annotation and predicted cell type labels from the PBMC dataset in Azimuth using FindTransferAnchors and TransferData (Seurat). Marker gene statistics were calculated using wilcoxauc (presto). Genes consistently increased following 24c5 SEHFST LS antibody treatment were defined as those: (i) with a Mann-Whitney p-value < 0.1 and a log2 fold change greater than 0.25 compared to either the 24c5 IgG1 or no Ab condition, (ii) detected in a minimum fraction of 0.1 cells in either of the two conditions, and (iii) with a log2 fold change greater than 0 compared to both the 24c5 IgG1 and no Ab conditions. Genes consistently decreased following 24c5 SEHFST LS antibody treatment were defined as those: (i)with a Mann-Whitney p-value < 0.1 and a log2 fold change less than -0.25 compared to either the 24c5 IgG1 or no Ab conditions, (ii) detected in a minimum fraction of 0.1 cells in either of the two conditions, and (iii) a log2 fold change less than 0 compared to both the 24c5 IgG1 and no Ab conditions.
Assembly: GRCh38
Supplementary files format and content: Tab separated values files and original H5 outputs
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| Submission date |
Jun 28, 2024 |
| Last update date |
Jul 01, 2024 |
| Contact name |
Edward B Irvine |
| E-mail(s) |
edward.irvine@bsse.ethz.ch
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| Organization name |
Ragon Institute of MGH, MIT, and Harvard
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| Street address |
400 Technology Square
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE271079 |
Fc-engineered antibodies leverage neutrophils to drive control of Mycobacterium tuberculosis |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM8368288_Lane1_filtered_barcodes.tsv.gz |
56.6 Kb |
(ftp)(http) |
TSV |
| GSM8368288_Lane1_filtered_feature_bc_matrix.h5 |
45.0 Mb |
(ftp)(http) |
H5 |
| GSM8368288_Lane1_filtered_features.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM8368288_Lane1_raw_barcodes.tsv.gz |
6.5 Mb |
(ftp)(http) |
TSV |
| GSM8368288_Lane1_raw_feature_bc_matrix.h5 |
72.5 Mb |
(ftp)(http) |
H5 |
| GSM8368288_Lane1_raw_features.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| Raw data not provided for this record |
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