Journal: Nature

Article Title: Decoding human fetal liver haematopoiesis

doi: 10.1038/s41586-019-1652-y

Figure Lengend Snippet: a, Schematic of tissue processing and cell isolation for scRNA-seq profiling of fetal liver, skin and kidney across four developmental stages (7-8, 9-11, 12-14, and 15-17 post conception weeks (PCW)), and yolk sac from 4-7 PCW. SS2, Smart-seq2. b, UMAP visualisation of fetal liver cells from 10x using 3’ chemistry. Colours indicate cell state. HSC/MPP, haematopoietic stem cell/multipotent progenitor; ILC, innate lymphoid cell; NK, natural killer cell; Neut-myeloid, neutrophil-myeloid; DC, dendritic cell; pDC, plasmacytoid DC; Mono-mac, monocyte-macrophage; EI, erythroblastic island; Early L/TL, Early lymphoid/T lymphocyte; MEMP, megakaryocyte-erythroid-mast cell progenitor. Statistical significance of cell frequency change by stage shown in parentheses (negative binomial regression with bootstrap correction for sort gates; * p < 0.05, *** p < 0.001, and **** p < 0.0001 as per ) with up/down arrows to indicate positive/negative coefficient of change, respectively. c , Liver composition by developmental stage as the mean percentage of each population per stage corrected by CD45 + /CD45 - sort fraction. Colours indicate cell states as shown in b.

Article Snippet: We FACS-isolated CD45 + and CD45 - cells using adjoining gates for comprehensive capture ( and ) for single cell RNA-sequencing (scRNA-seq) (both 10x Genomics platform Smart-seq2) ( , , and ).

Techniques: Cell Isolation

Journal: Nature

Article Title: Decoding human fetal liver haematopoiesis

doi: 10.1038/s41586-019-1652-y

Figure Lengend Snippet: a , Fetal skin and kidney haematopoietic cells visualised by UMAP. Colours indicate cell state. Inset: colours indicate tissue type. b , UMAP visualisation of yolk sac haematopoietic cells. Colours indicate cell state. Inset: colours indicate location within yolk sac. c , UMAP visualisation of 3’ liver 10x cells post batch correction, coloured by sample. d , UMAP visualisation (top) of 3’ 10x liver sample sex mixing grouped by developmental stage, and violin plots (bottom) showing ln-normalised median expression of XIST (green) and RSP4Y1 (purple), which marks female and male samples respectively. e , UMAP visualisation of fetal liver composition by developmental stage. Colours indicate cell state. f, UMAP visualisation of fetal liver cells profiled using Smart-seq2. Colours indicate cell states as shown in e . g , Frequency (mean +/- s.e.m.) of B cells in the CD34 - cells detected in 6-19 PCW fetal livers by flow cytometry (* p < 0.05; *** p = 0.003; **** p < 0.001).

Article Snippet: We FACS-isolated CD45 + and CD45 - cells using adjoining gates for comprehensive capture ( and ) for single cell RNA-sequencing (scRNA-seq) (both 10x Genomics platform Smart-seq2) ( , , and ).

Techniques: Expressing, Flow Cytometry

Journal: Nature

Article Title: Decoding human fetal liver haematopoiesis

doi: 10.1038/s41586-019-1652-y

Figure Lengend Snippet: a, Gating strategy used to FACS-isolate cells for droplet-(10x) and plate-based scRNA-seq (Smart-seq2) for samples F2-F17. b, Gating strategy used to FACS-isolate cells for cytospins, scRNA-seq (Smart-seq2) and 100 cell RNA-seq. c, Flow cytometry gating strategy used to identify the colonies cultured in vitro from single cells as shown in , Flow cytometry gating strategy used to identify B and NK colonies cultured in vitro from 10 cells as shown in .

Article Snippet: We FACS-isolated CD45 + and CD45 - cells using adjoining gates for comprehensive capture ( and ) for single cell RNA-sequencing (scRNA-seq) (both 10x Genomics platform Smart-seq2) ( , , and ).

Techniques: RNA Sequencing, Flow Cytometry, Cell Culture, In Vitro

Journal: Nature

Article Title: Decoding human fetal liver haematopoiesis

doi: 10.1038/s41586-019-1652-y

Figure Lengend Snippet:

Article Snippet: We FACS-isolated CD45 + and CD45 - cells using adjoining gates for comprehensive capture ( and ) for single cell RNA-sequencing (scRNA-seq) (both 10x Genomics platform Smart-seq2) ( , , and ).

Techniques:

Journal: Science immunology

Article Title: Airway Brush Cells Generate Cysteinyl Leukotrienes Through the ATP Sensor P2Y2

doi: 10.1126/sciimmunol.aax7224

Figure Lengend Snippet: EpCAM+CD45−/loweGFP+ and EpCAM+CD45−/loweGFP− EpCs were isolated by FACS sorting from naïve nasal mucosa of ChAT-eGFP mice. (A) FACS gating strategy demonstrating two populations of nasal ChAT-eGFP+ EpCs. (B) ChAT-eGFP+ EpCs recovered as a percent of all live cells (left) and total number recovered per mouse (middle). Frequency of FSChiSSChi and FSClowSSClow ChAT-eGFP+ EpCs as a percent of all ChAT-eGFP+ EpCs (right). (C, E). Normalized counts of the indicated genes derived from RNA-seq analysis using DeSeq2. (D) Hierarchical clustering of the top 50 most variably expressed genes among the ChAT-eGFP+ EpCs in the trachea and nose. (F) Whole mount of nasal septum of a ChAT-eGFP mouse, eGFP fluorescence was enhanced with an anti-eGFP antibody (green). (G) Whole nasal septum staining for DCLK1 (red), ChAT-eGFP (green) and EpCAM (grey). (H) Cross-section of the nasal cavity and staining of paraffin embedded slides for ChAT-eGFP (green) , Gα-gustducin (red) and Hoechst (blue).

Article Snippet: Low input RNA sequencing Cells from nasal single cell suspensions were processed at the Broad Institute Technology Labs using low-input eukaryotic Smart-seq 2.

Techniques: Isolation, Derivative Assay, RNA Sequencing, Fluorescence, Staining

Journal: Science immunology

Article Title: Airway Brush Cells Generate Cysteinyl Leukotrienes Through the ATP Sensor P2Y2

doi: 10.1126/sciimmunol.aax7224

Figure Lengend Snippet: (A) Principal component analysis of ChAT-eGFP+ BrCs and ChAT-eGFP− EpCs from the nose and trachea using the top 100 most variable transcripts. Numbers indicate frequency of transcripts described by each principal component. (B) Euclidean distance matrix of the rlog transformed values derived from RNA sequencing. (C) Hierarchical clustering of the top 100 most variable genes derived from DeSeq2 analysis. Transcripts of enzymes in the CysLT biosynthetic pathway are highlighted in red. Transcripts of proteins in the phosphatidylinositol pathway are highlighted in green. (D) Normalized counts of transcripts encoding CysLT biosynthetic enzymes and transporters. (E) BrCs (EpCAM+CD45low/−eGFP+), CD45+ cells and EpCs (EpCAM+CD45−eGFP−) were isolated from the nasal mucosa and stimulated ex vivo with the indicated doses of calcium ionophore (A23187). Where indicated, cells were pre-treated for 15 min with the FLAP inhibitor MK-886. The concentration of CysLTs in the supernatants was measured by ELISA at 30 min. Data are means ± SEM, from at least three independent experiments, each dot represents a separate biological replicate, * p<0.05, ** p<0.01.

Article Snippet: Low input RNA sequencing Cells from nasal single cell suspensions were processed at the Broad Institute Technology Labs using low-input eukaryotic Smart-seq 2.

Techniques: Transformation Assay, Derivative Assay, RNA Sequencing, Isolation, Ex Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay