Gene Expression
Nebulas
Gene Expression NebulasSummary: Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the sub-capsular sinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.
Overall Design: Droplet-based single-cell RNA sequencing (scRNAseq) of human CD45- CD31+ PDPN+ cells from the axillary LNs (AXLNs) and head and neck LNs (HNLNs). ScRNAseq was performed using 10X Genomics Single Cell 3' solution, version 2 according to manufacture's instructions. Libraries were sequenced on HiSeq3000.
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| Treatment Protocol: | - |
| Extract Protocol: | Immediately after surgery, human LNs were transferred into RPMI on ice and cut into small pieces, and digested for 1h with RPMI containing collagenase P, dispase and DNase. Hematopoietic cells were depleted with EasySepTM Human CD45 Depletion Kit (Stem Cell Technologies), and the single cell suspension was incubated with PE-anti-PDPN, AF488-anti-CD45, APC-anti-CD31 and LIVE/DEADTM Fixable Near-IR Dead Cell Stain kit (ThermoFisher Scientific, L10119) for 30 min. Live CD45- PDPN+ CD31+ cells known to include LECs were sorted with FACS Aria (BD Bioscience) or SH800S (Sony) into RPMI medium containing 10% FCS. Sorted cells were immediately count and run on the 10X Chromium (10X Genomics). |
| Library Construction Protocol: | scRNA-seq libraries were prepared according to the manufacturer's instructions (CG00052 RevB) using Chromium™.Single Cell 3' Library and Gel Bead Kit v2 (10X Genomics, 120237) and Chromium™ Single Cell A Chip Kit (10X Genomics, 120236). |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 3000 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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