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| Status |
Public on Oct 07, 2022 |
| Title |
mLN_day300_2 |
| Sample type |
SRA |
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| Source name |
gut-draining mesenteric lymph node
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| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
cell type: ex vivo isolated cells CD24-CD45-
tissue: gut-draining mesenteric lymph node
housing: specific pathogen free
time: day300
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| Treatment protocol |
For stromal cell isolation, mLNs (small intestinal and colon/caecum-draining) were resected and digested in RPMI 1640 medium (Gibco, Cat. #72400021) containing 0.2 mg/ml collagenase P (Roche, Cat. #11213865001), 0.15 U/ml dispase (Roche, Cat. #4942078001) and 0.2 mg/ml DNase I (Roche, Cat. #4536282001) as described previously (Malhotra et al., 2012). After digestion, cells were kept at 4 °C in PBS containing 0.2 % BSA (Merck, Cat. #A2058) and 5 mM EDTA (Roth, Cat. #8043.1). CD45- cells were enriched by autoMACS separation after magnetic labeling of CD45+ cells using anti‑CD45‑APC followed by anti‑APC microbeads (Miltenyi Biotec, Cat. #130-090-855) or anti-CD45 Nanobeads (Biolegend, Cat. #480028). Subsequently, the CD45- fraction was stained using fluorochrome-coupled antibodies and used to sort CD45‑CD24‑CD31‑gp38+ FSCs (Aria II, 100 μm nozzle) and bulk CD45‑CD24‑ non-hematopoietic cells (Aria III, 70 μm nozzle) for RNA‑seq/ATAC‑seq and scRNA‑seq, respectively.
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| Growth protocol |
Mice were housed under specific pathogen free conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single CD45‑CD24‑ cells were sorted by FACS ARIA III (BD) and collected in PBS containing 0.04 % w/v BSA at a density of 400 cells/μl. Chromium™ Controller was used for partitioning single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs)
Single Cell 3’ reagent kit v2 for reverse transcription, cDNA amplification and library construction (10xGenomics, Cat. #120236). The detailed protocol was provided by 10xGenomics. SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems). Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent).
Sequencing was performed in paired-end mode (2 x 75 cycles) using NextSeq 500 sequencer (Illumina) to attain approximately 75,000 +/- 25,000 reads per single cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
mLN_day300_2
RCM_mLN_day300_scRNAseq.zip
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| Data processing |
Data were demultiplexed using Cell Ranger software (version 2.0.0) based on 8 bp 10X sample indexes and paired-end FASTQ files generated. The cell barcodes and transcript UMIs were processed as previously described (Zheng et al., 2017). Subsequently, read 2, which contains the transcript insert sequence, was aligned to mouse UCSC mm10 reference genome using STAR aligner with default parameters (Dobin and Gingeras, 2015). The alignment results were used to quantify the expression level of mouse genes and generation of gene-barcode matrix, which were further processed with Seurat (version 2.3.3). All cells received an identifier which was used as common meta-data throughout the analysis including differentiation trajectories and dynamic gene regulatory networks (see below).
All cells with less than 1,000 or more than 4,600 detected genes per cell were filtered out. Moreover, cells with more than 4.5 % read mapping to mitochondrial genes were removed yielding 15,659 cells passing QC. All cells across all developmental stages were subjected to canonical correlation analysis and regressed against the variables, number of UMIs, percent of mitochondrial reads, percent of ribosomal reads and scores for the proliferation S.Score and G2M.Score computed with CellCycleScoring(). t‑SNE dimensionality reduction was performed using the first 12 dimensions of the CCA and resolution set to 1.1. Only clusters (non-endothelial MC) with expression for Pecam < 1 were used for the further analysis amounting to 9,323 cells which were re-embedded as described above (resolution = 1.0). The perivascular mesenchymal cell (PvMC) cluster and clusters classified as adjacent cells were excluded and the remainder cells, numbering 5,658 mesenchymal cells were re-embedded as described above (resolution = 1.0). For subsetting the Prog. subset, 259 cells were re-embedded as described above (resolution = 0.5, dimsuse = 10). Gene ontology (GO) analysis was performed for differentially upregulated genes per cluster using TopGO (Alexa and Rahnenfuhrer, 2010).
Differentiation trajectories were analyzed using Monocle (version 2.18.0) (Trapnell et al., 2014). Unsupervised ordering was performed on the 5,658 mesenchymal cells using Monocle2’s DDRTree algorithm based on genes with a mean expression >0.1. The trajectory containing all mesenchymal cells, was split based on marker gene expression Vcam1 and Cd34 and the annotation of cells belonging to the ProgCxcl13+ or ProgCD34+ subset to distinct terminal branches. Two separate unsupervised orderings were performed as described above and are referred to as CD34+SC or FRC trajectory.
Gene regulatory networks were inferred with the dynGENIE3 algorithm, where the input expression values were based on ordering the genes according to physiological age per branch for each of the two trajectories being CD34+SC or FRC (Huynh-Thu and Geurts, 2018). The list of candidate TFs was derived from TFBS from ATAC-seq profiling or DMRs within the proximity of TFs. For network visualization with Cytoscape (version 3.6.0)(Shannon et al., 2003), only the top 500 links ranked by weight assigned by dynGENIE3 were used. Node centrality and betweenness were calculated with the degree and betweenness functions from the igraph (version 1.2.2) package.
Genome_build: mm10
Supplementary_files_format_and_content: CellRanger processed fastq output yielding RMCs and gene annotation
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| Submission date |
Apr 21, 2021 |
| Last update date |
Oct 07, 2022 |
| Contact name |
Joern Pezoldt |
| E-mail(s) |
jorn.pezoldt@epfl.ch
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| Phone |
0041766040171
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| Organization name |
EPFL
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| Department |
SV
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| Lab |
Laboratory Systems Biology and Genetics
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| Street address |
Station 19, SV 3818.A
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE172514 |
Postnatal expansion of the lymph node stromal cell pool towards reticular and CD34+ stromal cell subsets [scRNA-seq] |
| GSE172526 |
Postnatal expansion of the lymph node stromal cell pool towards reticular and CD34+ stromal cell subsets |
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| Relations |
| BioSample |
SAMN18823090 |
| SRA |
SRX10653844 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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