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| Status |
Public on Dec 23, 2020 |
| Title |
active_KR71 |
| Sample type |
SRA |
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| Source name |
dematricized lung cells
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| Organism |
Macaca mulatta |
| Characteristics |
disease: active tuberculosis
infection strain: CDC1551
cfu delivered: 1000
celll type: dematricized lung cells
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| Treatment protocol |
All animal procedures were approved by the Tulane National Primate Research Center (TNPRC) Institutional Animal Care and Use Committee and performed in strict accordance with the NIH, USDA and AALAAC guidelines. Specific-pathogen-free, retrovirus-free, mycobacteria-naive, adult rhesus macaques were bred and housed at the TNPRC and they were assigned into three groups. One group of macaques (n=4) remained uninfected. The second (~ 10 CFU) (n=4) (LTBI) and third (~1000 CFU) group (n=6) (PTB) of animals were aerosol-challenged with Mtb CDC1551 using a custom head-only dynamic inhalation system housed within a class III biological safety cabinet as described extensively before (Bucsan et al., 2019a; Foreman et al., 2020; Foreman et al., 2016; Kaushal et al., 2015; Mehra et al., 2015). Humane endpoints were predefined in the animal use protocol and applied as a measure of reduction of discomfort. Criteria for euthanasia included presentation of four or more of the following conditions: (i) body temperatures consistently greater than 2°F compared to pre-infection values for 3 or more weeks in a row; (ii) loss of body weight for 15% or more; (iii) serum CRP values higher than 10 mg/mL for 3 or more consecutive weeks, since CRP is a marker for systemic inflammation and exhibits a high degree of correlation with active TB in macaques; (iv) Thoracic radiographs (CXR) values higher than 2 on a scale of 0–4; with a score of 0 denoting normal lung and a score of 4 denoting severe tuberculous pneumonia and pathology. (v) respiratory discomfort leading to vocalization; (vi) significant or complete loss of appetite; and (vii) detectable bacilli in BAL samples. At necropsy, lung, spleen, and liver tissues were collected and processed, as previously described, using two sections of pulmonary tissue that represented every lung lobe in at least one sample; CFU were determined per gram of tissue. We also evaluated CFU from individual lung granulomas isolated at necropsy, as described earlier.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At necropsy lung tissues were collected and digested with DNase I and Liberase at 37ºC for 30-45 mins. Digested lung tissue was gently dispersed by passage through a 70 μm pore size nylon tissue strainer (Falcon; BD Biosciences); the resultant single-cell suspension was treated with ACK lysis buffer to remove any residual RBC, washed twice, and counted, as previously described (Foreman et al., 2016; Gautam et al., 2018; Kaushal et al., 2015; Kuroda et al., 2018; Mehra et al., 2015). Cells prepared in this way were stored at -70ºC and then used for downstream processing of scRNA-seq.
scRNA-seq was done according to the manufacturer instructions (10X genomics). Briefly, after quickly thawing the frozen lung single cell suspension in water bath, 2X106 cells were taken for downstream processing. Cell suspensions were enriched for CD45 by using NHP CD45-microbeads according to the manufacturer instruction (Milteny Biotec). After then, CD45+ enriched lung single cell suspensions were subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 3’ (v3) Reagent Kit in the BSL-3 level laboratory as per manufacturer’s instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/μL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer’s instructions. Libraries were sequenced on a NovaSeq S4 (200 cycle) flow cell, targeting 50,000 read pairs/cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The Cell Ranger Single-Cell Software 3.0 available at 10X website was used to perform sample demultiplexing. We aligned resulting fastq files on mmul10 genome (Genebank, https://www.ncbi.nlm.nih.gov/assembly/GCF_003339765.1/, with addition of Ensembl mmul8 mitochondrial genes for GTF file) with cellranger count. For each sample the recovered-cells parameter was set to 10,000 cells that we expected to recover for each individual library. We used R package Seurat 3 for downstream analysis of count matrixes that we got as output from cellranger count. We filtered cells that (1) had more than 12.5% (samples KP77, KR71), 20% (samples KM67, KN08, LD09, LE99, 33935, KP87), 25% (samples GP50, HV02), 30% (samples KN21, 31810) of mitochondrial gene content, (2) had less than 725 detected genes. We chose different thresholds for mitochondrial gene content due to different distributions between samples. Data was log-normalized with a scale factor of 104. The most variable genes were detected by the FindVariableFeatures function and used for subsequent analysis. Latent variables (number of UMI’s and mitochondrial content) were regressed out using a negative binomial model (function ScaleData). Principle component analysis (PCA) was performed with RunPCA function. A UMAP dimensionality reduction was performed on the scaled matrix (with most variable genes only) using the first 20 PCA components to obtain a two-dimensional representation of the cell states. For clustering, we used the functions FindNeighbors (20 PCA) and FindClusters (resolution 0.25).
Genome_build: mmul10
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| Submission date |
May 02, 2020 |
| Last update date |
Dec 24, 2020 |
| Contact name |
Maxim N. Artyomov |
| E-mail(s) |
martyomov@pathology.wustl.edu
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| Organization name |
Washington University in St.Louis
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| Department |
Immunology&Pathology
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| Street address |
660 S. Euclid Avenue, Campus Box 8118
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| City |
St.Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL27943 |
| Series (1) |
| GSE149758 |
Defining the tuberculosis lung landscape during disease and latency using single cell technologies |
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| Relations |
| BioSample |
SAMN14816280 |
| SRA |
SRX8242037 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4511884_active_KR71_filtered_feature_bc_matrix.h5 |
3.3 Mb |
(ftp)(http) |
H5 |
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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