As a first pass to develop common Standard Operating Protocols to produce compatible single-cell RNAseq across the consortium, Immgen labs (Wash U. St Louis, Broad, HMS) generated parallel datasets from spleens of un-perturbed C57Bl/6 mice.
Per ImmGen SOP, mice were 5 w.o. C57Bl/6J (B6) mice bred at JAX, shipped one week prior and maintained in SPF conditions. Spleens were mechanically dissociated, and CD45+ cells were purified by flow cytometry immediately prior to encapsulation on a 10X genomics instrument. Libraries were constructed with the Chromium Single Cell 3’ Library Kit. After QC and selection of cells passing depth criteria (>250 genes/cell), 10651 cells from a B6 female donor are displayed.

As a first pass to develop common Standard Operating Protocols to produce compatible single-cell RNAseq across the consortium, Immgen labs (Wash U. St Louis, Broad, HMS) generated parallel datasets from spleens of un-perturbed C57Bl/6 mice.
Per ImmGen SOP, mice were 5 w.o. C57Bl/6J (B6) mice bred at JAX, shipped one week prior and maintained in SPF conditions. Spleens were mechanically dissociated, and CD45+ cells were purified by flow cytometry (Aria) immediately prior to encapsulation on a 10X genomics instrument. Libraries were constructed with the Chromium Single Cell 3’ Library Kit. After QC and selection of cells passing depth criteria (>250 genes/cell), we obtained valid results for 3025 and 2733 cells from female and male B6 donors, respectively.

CD4+ T regulatory cells (Treg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of Treg or conventional CD4+FoxP3- T cells (Tconv) from mouse lymphoid organs. Treg and Tconv pools showed areas of overlap, as resting 'furtive' Tregs (category 1) with overall similarity to Tconvs or as a convergence of activated states (categories: 2/3/4).
To better understand the phenotypic heterogeneity of CD4+ regulatory T cells (Treg) we generated single-cell RNAseq data using the InDrops protocol. Spleens were harvested from 6-8 week old male Foxp3ires-gfp/B6 mice maintained at HMS. Spleens were mechanically dissociated to single cell suspensions. FoxP3+ Tregs (DAPI- TCRb+ CD4+ GFP+) and FoxP3- Tconv (DAPI- TCRb+ CD4+ GFP-) cells were sorted by flow cytometry. Tregs and Tconvs were encapsulated separately in nanodroplets according to the InDrops protocol.

Zemmour et al, 2018
Data availability: GEO: GSE110547

CD4+ T effector cells lymphocytes (Teff) are functionally heterogeneous, and traditionally divided into subsets defined by the cytokines they produce. To determine the actual states that Teff adopt in frontline tissues in vivo, we applied single-cell RNAseq on colonic Teff in a range of conditions: germfree, unchallenged Specific Pathogen Free, or after bacterial infection with Citrobacter rodentium or attenuated Salmonella typhimurium, or two helminths Heligmosomoides polygyrus and Nippostrongylus brasiliensis . We chose infection times that allowed responses to develop fully and achieve full Teff bias (day 11-13), and the pathogens elicited the expected biased cytokine responses. CD4+ T cells from control or infected mice were hashtagged with DNA-coded antibodies and comingled for sorting, 10X Genomics and library construction.

Bone marrow of un-perturbed C57Bl/6 mice for use in single-cell RNAseq analysis. Per ImmGen SOP, two mice 7 w.o. C57Bl/6J (B6) bred at JAX and shipped to HMS one week prior were maintained in SPF conditions. Bone marrow from one male and one female mouse was flushed using a 1mL syringe and 21G needle. Lineage-negative cells (Ter-119, Cd11b, Cd45R/B220, and Ly6G/Ly6C(Gr-1)) were purified by flow cytometry and encapsulated on a 10X genomics instrument with the Chromium Single Cell 3 Library kit.

Neutrophils mediate an expanding range of homeostatic and pathologic functions. Correspondingly they exhibit extensive phenotypic heterogeneity, but bona fide neutrophil subsets remain controversial. Here we applied single-cell RNA sequencing to >17,000 healthy and perturbed murine neutrophils across biological tissues. Using diffusion maps and RNA velocity analysis, we could instead model gene expression along a single chronologically-ordered developmental spectrum, “neutrotime”. Neutrotime extends from immature Ly6ghigh Camphigh LtfhighLcn2high neutrophils, found in greatest abundance in the bone marrow, to Il1bhigh Ccl6high Csf3rhigh neutrophils that predominate in blood and spleen. Data from developing neutrophils and ex vivo-differentiated neutrophils integrated smoothly into neutrotime. Experimental inflammation of lung, peritoneum and joint translated into de novo transcriptional activity reflecting structured permutations of the neutrotime paradigm.
Authors: Grieshaber-Bouyer R, Radtke F, Cunin P, Stifano G, Levescot A, Vijaykumar B, Nelson-Maney N, Blaustein RB, Monach PA, Nigrovic PA, ImmGen Consortium

Thymi were extracted from C57Bl/6J mice (11-13 weeks old, 2 males, 2 females), and dissociated with collagenase D to maximally release lymphoid and non-lymphoid cell-types. Each thymus was stained (CD45, CD3e, CD4, CD8, DNA-tagged barcodes – each mouse tagged individually) per Immgen SOP Thymocytes were sorted to rebalance subpopulations in the final sample (20% total CD45+ thymocytes, 47% CD45+CD3hi, and 33% CD45+CD4-CD8- double negatives). The sorted cells were immediately taken for encapsulation and single-cell RNAseq using the 10x 3’v3.1 protocol.

Lungs were harvested from naive C57BL/6 mice (8-12 weeks, male). The lungs were digested with collagenase IV. Monocytes and macrophages were sorted as CD45+ CD3-CD19-NKP46- CD11b+LY6G- and encapsulated using the 10X Chromium 3’ v2. This study contains two data sets from naive mouse lungs. One data set (GSM4877984_BM) contains bone marrow-derived monocytes and macrophages while the other data set (GSM4773604) contains non-bone marrow-derived monocytes and macrophages.
Casanova-Acebes et al., Nature (2021).

Lungs and lymph nodes were harvested from C57Bl/6J mice (male, 12 weeks). The tissues were digested with collagenase IV and sorted (CD45+, lin-, MHCII+, CD11c+). The sorted cells were taken for encapsulation and single-cell RNAseq using the 10x Chromium platform.
Maier et al, 2020

Spleen, blood, IELs, kidney, salivary gland, fat and liver were harvested from CD8a-APCFlour780 IV injected C57BL/6J mice (6-8 weeks, male and female), 32 days post viral infection. Spleen and blood were treated with ACK lysis buffer. Kidneys, salivary gland, liver, and fat were digested with collagenase. Lymphocytes from the small intestine, kidney, salivary gland, and liver were separated on a 44%/67% Percoll density gradient. P14 cells were sorted from spleen and blood. IV- P14 cells were sorted from kidney, salivary gland, IELs, liver, and fat. The sorted cells were taken for encapsulation and RNAseq using the 10x Chromium platform.
Crowl et al, 2022