Droplet-based Single-cell RNA Sequencing Research Articles

Pi-seq—A customizable multichannel syringe pump for microfluidics

The advent of single cell technologies resulted in growing demand for microfluidics in the biological sciences. Commercial platforms have remained expensive, inflexible, and non-customizable black boxes. We developed an open source, multichannel, zero-backflow microfluidics device based on syringe pumps controlled by a Raspberry Pi computer. It uses both readily available and 3D-printed parts as well as a custom PCB and is easily serviceable. Moreover, it is fully customizable for various applications. Total cost is under €600. We equipped one channel with a custom Peltier-based temperature controller for precise heating or cooling and a mixer mechanism to prevent sedimentation of the cells within the syringe. Depending on the cells in the sample, heating and cooling can be useful to maintain a beneficial environment or to slow down cellular processes and cell death, respectively. Combined with microfluidics consumables and a microscope, the device is capable of integration into a high quality droplet-based single cell RNA sequencing workflow as shown here. Analysis of a mixture of human and insect cells resulted in a dataset of 17,769 single cells and demonstrates reliable operation and separation.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

Single-cell RNA sequencing reveals characteristics of myeloid cells in post-acute sequelae of SARS-CoV-2 patients with persistent respiratory symptoms.

Although our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complications in COVID-19 infection remains unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequelae of SARS-CoV-2 infection (PASC) with persistent pulmonary symptoms. The dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. To characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing (scRNA-seq) to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from a participant naïve to SARS-CoV-2 (Control) (n=1) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC) (n=2). After integrating scRNA-seq data with a naïve participant from a published dataset, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14+/CD16+monocytes, and dendritic cells) was increased in PPASC (n=2) compared to controls (n=2). MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis-related (VEGF, WNT, and SMAD) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. Further comparison of PPASC with scRNA-seq data with Severe COVID-19 (n=4) data demonstrated enrichment of fibrotic transcriptional signatures. In PPASC, we observed interactive VEGF ligand-receptor pairs among MLCs, and network modules in CD14+ (cluster 4) and CD16+ (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID-19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS-CoV-2 naïve samples. Analysis of a small scRNA-seq dataset demonstrated alterations in the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests a potential role in PPASC development.

SEVtras delineates small extracellular vesicles at droplet resolution from single-cell transcriptomes.

Small extracellular vesicles (sEVs) are emerging as pivotal players in a wide range of physiological and pathological processes. However, a pressing challenge has been the lack of high-throughput techniques capable of unraveling the intricate heterogeneity of sEVs and decoding the underlying cellular behaviors governing sEV secretion. Here we leverage droplet-based single-cell RNA sequencing (scRNA-seq) and introduce an algorithm, SEVtras, to identify sEV-containing droplets and estimate the sEV secretion activity (ESAI) of individual cells. Through extensive validations on both simulated and real datasets, we demonstrate SEVtras' efficacy in capturing sEV-containing droplets and characterizing the secretion activity of specific cell types. By applying SEVtras to four tumor scRNA-seq datasets, we further illustrate that the ESAI can serve as a potent indicator of tumor progression, particularly in the early stages. With the increasing importance and availability of scRNA-seq datasets, SEVtras holds promise in offering valuable extracellular insights into the cell heterogeneity.

Loss of the OX40 Target in Lymphoma Patients after Combining an Anti-OX40 Agonist Antibody with in Situ Vaccination

INTRODUCTION:Previous clinical trials have shown that in situ vaccination using the combination of low-dose radiotherapy and intratumoral injection of CpG, a Toll-like receptor 9 (TLR9) agonist, can elicit T cell immune responses in patients with low grade lymphoma and regression at both the treated and non-treated distal tumor sites in patients with low-grade lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discovery 2018, Hammerich et al., Nat Med 2019). OX40 is a T cell activating co-receptor, belonging to the TNFR superfamily. Ligation of this receptor by an agonist anti-OX40 antibody leads to enhanced T cell activation of effector CD4 T cells while inhibiting Treg function. In preclinical models, addition of an anti-OX40 antibody to intratumoral CpG cured established tumors (Sagiv-Barfi I, et al. Sci Transl Med. 2018). Intratumoral injection of CpG up-regulated OX40 on CD4 T cells in the microenvironment enhancing the binding of agonistic anti-OX40 antibody that triggered a robust immune response. These results provided the rationale for a clinical trial of in situ vaccination (NCT03410901) using a human IgG1 agonistic anti-OX40 antibody (BMS-986178). STUDY:Fourteen patients with low-grade lymphoma received low-dose (2Gy x 2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 2 mg CpG-ODN (SD-101, TriSalus Life Sciences) and 3.75 mg anti-OX40 antibody into the same site. 40mg of anti-OX40 was administered intravenously starting on day 2 and continuing every 4 weeks for a total of 6 infusions. Therapy was well tolerated with no dose-limiting toxicities. Response to therapy was assessed using the revised Lugano criteria (Cheson et al., JCO 2014) based on CT scans at 12, 24, 48, 72, and 96 weeks. Fine needle aspirates (FNA) were performed at both the injected and a non-injected site outside the radiation field prior to, and at 1 and 6 weeks after initiation of treatment. Cells were analyzed by flow cytometry and droplet-based single-cell RNA sequencing to determine population dynamics, response to therapy as well as antibody binding and target occupancy by the anti-OX40 antibody. RESULTS:Of the 14 patients enrolled, 1 patient attained a partial response (PR), while 9 had stable disease (SD) and 4 had disease progression (PD) as their best overall response. Investigation into the binding of the treatment antibody and target occupancy by flow cytometry showed saturation of all the OX40 sites on CD4 T cells by week 6 at both the injected and non-injected tumors. However, further investigation also showed there was significantly less OX40 on the cell surface after treatment, suggesting a loss of the antibody target (Figure 1). This loss was reproduced by in vitro assays where activated blood CD4 T cells exhibited a loss of cell surface OX40 when cultured with BMS-986178. Additionally, Quigley et al ( Clin Cancer Res 2019) previously showed that an OX40 receptor occupancy of greater than 40% led to a loss of target in mice and in blood T cells of solid tumor patients. We further observed that the loss of cell surface OX40 occurred in all CD4 T subsets, including effector CD4 T cells and T Regulatory cells (Tregs). This result contrasted with our in vivo and in vitro findings in successful therapeutic mouse models where we did not observe this loss of cell surface OX40 on effector CD4 T cells after treatment with a mouse-specific anti-OX40 agonist antibody. C ONCLUSION: Our observations suggest that treatment with an agonistic anti-OX40 antibody induced a loss of OX40 on the cell surface of effector CD4 T cells in the tumor microenvironment. Fewer molecules of OX40 receptor may have constrained the efficacy of subsequent anti-OX40 infusions and may explain why the clinical responses observed in this study were lower than observed in our past clinical trials of in situ vaccination.

A targeted sequencing extension for transcript genotyping in single-cell transcriptomics.

As no existing methods within the single-cell RNA sequencing repertoire combine genotyping of specific genomic loci with high throughput, we evaluated a straightforward, targeted sequencing approach as an extension to high-throughput droplet-based single-cell RNA sequencing. Overlaying standard gene expression data with transcript level genotype information provides a strategy to study the impact of genetic variants. Here, we describe this targeted sequencing extension, explain how to process the data and evaluate how technical parameters such as amount of input cDNA, number of amplification rounds, and sequencing depth influence the number of transcripts detected. Finally, we demonstrate how targeted sequencing can be used in two contexts: (1) simultaneous investigation of the presence of a somatic variant and its potential impact on the transcriptome of affected cells and (2) evaluation of allele-specific expression of a germline variant in ad hoc cell subsets. Through these and other comparable applications, our targeted sequencing extension has the potential to improve our understanding of functional effects caused by genetic variation.

Demuxmix: Demultiplexing oligonucleotide-barcoded single-cell RNA sequencing data with regression mixture models.

Droplet-based single-cell RNA sequencing (scRNA-seq) is widely used in biomedical research for interrogating the transcriptomes of single cells on a large scale. Pooling and processing cells from different samples together can reduce costs and batch effects. To pool cells, they are often first labeled with hashtag oligonucleotides (HTOs). These HTOs are sequenced alongside the cells' RNA in the droplets and subsequently used to computationally assign each droplet to its sample of origin, a process referred to as demultiplexing. Accurate demultiplexing is crucial but can be challenging due to background HTOs, low-quality cells/cell debris, and multiplets. A new demultiplexing method based on negative binomial regression mixture models is introduced. The method, called demuxmix, implements two significant improvements. First, demuxmix's probabilistic classification framework provides error probabilities for droplet assignments that can be used to discard uncertain droplets and inform about the quality of the HTO data and the success of the demultiplexing process. Second, demuxmix utilizes the positive association between detected genes in the RNA library and HTO counts to explain parts of the variance in the HTO data resulting in improved droplet assignments. The improved performance of demuxmix compared to existing demultiplexing methods is assessed using real and simulated data. Finally, the feasibility of accurately demultiplexing experimental designs where non-labeled cells are pooled with labeled cells is demonstrated. R/Bioconductor package demuxmix (https://doi.org/doi:10.18129/B9.bioc.demuxmix). Supplementary data are available at Bioinformatics online.

Transcriptional and spatial profiling of the kidney allograft unravels a central role for FcyRIII+ innate immune cells in rejection

Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the cell-to-cell interactions of the alloreactive inflammatory infiltrate. Here, we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies with varying phenotypes and severities of rejection and without rejection, and identified cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A+ NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A+ NK cells overexpressed FCRL3, suggesting that antibody-dependent cytotoxicity is a central mechanism of NK-cell mediated graft injury. Multiplexed immunofluorescence using 38 markers on 18 independent biopsy slides confirmed this role of FcγRIII+ NK and FcγRIII+ nonclassical monocytes in antibody-mediated rejection, with specificity to the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and identify several potential therapeutic targets that might improve allograft longevity.

LACK OF SMARCB1 EXPRESSION CHARACTERIZES A SUBSET OF PERIPHERAL T‐CELL LYMPHOMAS ENRICHED IN CHILDREN AND YOUNG ADULTS

Introduction: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of malignancies with poor outcome. Because patients respond poorly to current treatment regimens, identification of new therapeutic strategies is required. In a mouse tumor model, conditional deletion of Smarcb1 encoding a member of the SWI/SNF chromatin remodeling complex causes the majority of mice to develop mature T-cell lymphomas. The human counterpart, SMARCB1, is a bona fide tumor suppressor gene associated with the development of for example, rhabdoid tumors and schwannomas, but has also been shown to be involved in lymphocyte development. Methods: SMARCB1 (INI1) gene and protein expression was assessed in 315 patients with diverse mature T cell lymphomas by array-based mRNA profiling, immunohistochemistry and/or Western Blot. The DNA methylome was analyzed in SMARCB1-negative pediatric PTCL-NOS patients (n = 5) and murine T cell lymphomas of the Cd4-cre::Smarcb1fl/fl model (n = 5) using the Infinium MethylationEPIC and Mouse Methylation BeadChip, respectively. Murine Smarcb1-deficient T-cell lymphomas were further studied using droplet-based single cell RNA sequencing (Chromium, 10X Genomics). Results: SMARCB1 expression was heterogeneous between and within the 315 different human mature T-cell lymphoma entities. Loss of SMARCB1 expression in PTCL-NOS patients correlated with young age (Wald test, p value = 0.011). Molecular characterization and DNA methylation analysis revealed that loss of SMARCB1 expression in human PTCL largely occurs via somatic mutation and/or epigenetic silencing, whereas in contrast to rhabdoid tumors, germline SMARCB1 mutations were not observed. Comparison of the DNA methylome of human and murine PTCL-NOSSmarcb1- showed similar DNA methylation profiles, with hypermethylation of T-cell-related genes and hypomethylation of genes involved in myeloid development. Increase of myeloid cell populations was confirmed in murine tumors by scRNA-seq analyses, which further revealed an immunosuppressive and pro-inflammatory tumor microenvironment (TME). Treatment of tumor-bearing mice with SAHA, a pan-HDACi, triggered remodeling of the TME, promoting replenishment of lymphoid compartments and reversion of the exhaustion phenotype. Conclusions: Here we describe SMARCB1-negative PTCL-NOS as a potential new molecular subtype of PTCL-NOS significantly enriched in young patients. A strong concordance between naturally occurring SMARCB1-deficient PTCL in humans and in the targeted mouse model were found regarding epigenetic features. Our results provide a rationale for further investigation of potential HDACi combination therapies in SMARCB1-negative PTCL-NOS. Keywords: aggressive T-cell non-Hodgkin lymphoma, genomics, epigenomics, and other-omics No conflicts of interests pertinent to the abstract.

Diverse intratumoral heterogeneity and immune microenvironment of two HPV-related cervical cancer types revealed by single-cell RNA sequencing.

Cervical squamous cell carcinoma (SCC) and adenocarcinoma (AD) are the main histological types of human papillomavirus-related cervical cancer. However, there are few reports on cell type-specific molecular differences between SCC and AD. Here, we used unbiased droplet-based single-cell RNA sequencing to elucidate the cellular differences between SCC and AD in tumor heterogeneity, and tumor microenvironment (TME). A total of 61 723 cells from three SCC and three AD patients, were collected and divided into nine cell types. Epithelial cells exhibited high intra- and interpatient heterogeneity and functional diversity. Signaling pathways, such as epithelial-to-mesenchymal-transition (EMT), hypoxia and inflammatory response were upregulated in SCC, while cell cycle-related signaling pathways were highly enriched in AD. SCC was associated with high infiltration of cytotoxicity CD8 T, effector memory CD8 T, proliferativenatural killer (NK), and CD160+ NK cells as well as tumor-associated macrophages (TAMs) with high major histocompatibility complex-II genes. AD exhibited a high proportion of naive CD8 T, naive CD4 T, Treg CD4, central memory CD8, and TAMs with immunomodulatory functions. Additionally, we also observed that the majority of cancer-associated fibroblasts (CAFs) were from AD, and participated in inflammation regulation, while SCC-derived CAFs exhibited similar functions to tumor cells, such as EMT and hypoxia. This study revealed the widespread reprogramming of multiple cell populations in SCC and AD, dissected the cellular heterogeneity and characteristics in TME, and proposed potential therapeutic strategies for CC, such as targeted therapy and immunotherapy.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Single Cell RNA-Sequencing in Arabidopsis Root Tissues.

Droplet-based single-cell RNA-sequencing (scRNA-seq) empowers transcriptomic profiling with an unprecedented resolution, facilitating insights into the cellular heterogeneity of tissues, developmental progressions, stress-response dynamics, and more at single-cell level. In this chapter, we describe the experimental workflow of processing Arabidopsis root tissue into protoplasts and generating single-cell transcriptomes. We also describe the general computational workflow of visualizing and utilizing scRNA-seq data. This protocol can be used as a starting point for establishing a scRNA-seq workflow.

Single-Cell Transcriptomic Profiling of Müller Glia in the rd10 Retina.

Müller glia are the principal macroglia of the retina and support retinal neurons both in health and disease. In retinitis pigmentosa (RP), a highly heterogeneous inherited retinal disorder, the most common form of pathology involves primary rod degeneration, followed by secondary cone death. To investigate Müller glia responses to rod degeneration, we performed droplet-based single-cell RNA sequencing in the rd10 mouse model of RP during primary rod degeneration. We confirmed known MG behavior on gliosis, metabolic, and immune functions. Pde6brd10 Müller glia also exhibited an increased expression of histocompatibility complex members, which might arise from a novel immune function of Müller glia in RP. We also describe a possible decrease in glial lipid biogenesis, which might affect degenerating photoreceptors.

Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain.

Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells (PSCs) for application in disease modeling, diagnostics, drug screening, and cell-based therapies for Parkinson's Disease (PD). An increased understanding of the timing and molecular mechanisms promoting the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from PSCs. Here, we used droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach revealed that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons, which can be used to guide stem cell-based therapies and disease modeling approaches in PD.

7P HIFA1 inhibition as a therapeutic strategy for overcoming castration resistance in PTEN-deficient prostate cancer

Prostate cancer (PCa) is the second most commonly diagnosed neoplasia in men worldwide. The growth of prostate cancer cells is initially dependent of the activity of the androgen receptor (AR) and AR is the target of first-line prostate cancer systemic treatments. The benefit of adding neoadjuvant and adjuvant androgen deprivation therapy is well established for men with high-risk localized and advanced disease. However, some patients develop castration resistant prostate cancer, for which curative options are limited. PTEN is the most often tumor suppressor gene mutated in PCa patients. Hypoxic regions have been found in prostate adenocarcinoma, and elevated expression of hypoxia markers, including HIF1a (hypoxia-inducible factor 1A), have been shown to identify PCa patients with elevated risk of biochemical recurrence. Prostate tumors of sham-operated and castrated Pten(i)pe-/- mice, which have a prostatic luminal cell-specific inactivation of PTEN after puberty, were analyzed by droplet-based single cell-RNA sequencing. Moreover, the impact of genetic and pharmacological Hif1a inhibition was determined in castrated Pten(i)pe-/- mice by histological analyses. To gain insights on pathways driving androgen-independence in prostate tumor, we analyzed Pten(i)pe-/- mice which develop tumors that are resistant to castration. Droplet based single cell-RNA sequencing of 17265 cells from dissociated tumors of castrated and sham-operated Pten(i)pe-/- mice revealed that androgen deprivation enhances hypoxia signaling in luminal cells. Importantly, this is accompanied by the emergence of an immune-cell-related gene signature in luminal cells of castrated mice, indicating that activation of hypoxic signaling and induction of cellular plasticity are involved in castration resistance. Importantly, genetic ablation of Hif1a in prostatic tumors sensitizes them to castration, and pharmacological inhibition of Hif1a together with androgen deprivation leads to durable anti-tumoral response. Androgen deprivation in combination with HIF1 signaling inhibition is a promising therapeutic strategy for overcoming castration resistance in PTEN-deficient prostatic tumors.

Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues.

Brain-immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.

Scalable single-cell RNA sequencing from full transcripts with Smart-seq3xpress

Current single-cell RNA sequencing (scRNA-seq) methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity. Here we describe Smart-seq3xpress, which miniaturizes and streamlines the Smart-seq3 protocol to substantially reduce reagent use and increase cellular throughput. Smart-seq3xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete with common and rare cell types. Compared with droplet-based single-cell RNA sequencing that sequences RNA ends, the additional full-transcript coverage revealed cell-type-associated isoform variation.

Single-cell RNA sequencing of the retina in a model of retinitis pigmentosa reveals early responses to degeneration in rods and cones

BackgroundIn inherited retinal disorders such as retinitis pigmentosa (RP), rod photoreceptor-specific mutations cause primary rod degeneration that is followed by secondary cone death and loss of high-acuity vision. Mechanistic studies of retinal degeneration are challenging because of retinal heterogeneity. Moreover, the detection of early cone responses to rod death is especially difficult due to the paucity of cones in the retina. To resolve heterogeneity in the degenerating retina and investigate events in both types of photoreceptors during primary rod degeneration, we utilized droplet-based single-cell RNA sequencing in an RP mouse model, rd10.ResultsUsing trajectory analysis, we defined two consecutive phases of rod degeneration at P21, characterized by the early transient upregulation of Egr1 and the later induction of Cebpd. EGR1 was the transcription factor most significantly associated with the promoters of differentially regulated genes in Egr1-positive rods in silico. Silencing Egr1 affected the expression levels of two of these genes in vitro. Degenerating rods exhibited changes associated with metabolism, neuroprotection, and modifications to synapses and microtubules. Egr1 was also the most strongly upregulated transcript in cones. Its upregulation in cones accompanied potential early respiratory dysfunction and changes in signaling pathways. The expression pattern of EGR1 in the retina was dynamic during degeneration, with a transient increase of EGR1 immunoreactivity in both rods and cones during the early stages of their degenerative processes.ConclusionOur results identify early and late changes in degenerating rd10 rod photoreceptors and reveal early responses to rod degeneration in cones not expressing the disease-causing mutation, pointing to mechanisms relevant for secondary cone degeneration. In addition, our data implicate EGR1 as a potential key regulator of early degenerative events in rods and cones, providing a potential broad target for modulating photoreceptor degeneration.

Distinct Endothelial Cell Populations Characterize the Pulmonary Endarterectomy Specimen in Chronic Thromboembolic Pulmonary Hypertension

<h3>Purpose</h3> Misguided vascular repair is thought to contribute to the pathophysiology of chronic thromboembolic pulmonary hypertension (CTEPH), yet little is known about endothelial cell (EC) phenotypes in the organized thromboembolic material of patients with CTEPH. Therefore, we sought to characterize ECs from pulmonary endarterectomy (PEA) specimens. <h3>Methods</h3> Human PEA specimens from proximal pulmonary arteries were collected from five CTEPH patients and dissociated for droplet-based single-cell RNA sequencing. Data from three control human lungs was obtained from the Human Lung Cell Atlas (https://hlca.ds.czbiohub.org accessed Sept. 9, 2021). <h3>Results</h3> From the PEA specimens of five CTEPH patients, we obtained transcriptional profiles for 11068 cells representing 20 clusters and 9 cell types. From those, 4 clusters displayed markers of ECs. To identify appropriate control ECs, we assessed markers of endothelial subtype from the Human Lung Cell Atlas and identified markers of pulmonary and bronchial artery ECs. Therefore, we obtained the transcriptional profiles of pulmonary and bronchial artery ECs from the Human Lung Cell Atlas as controls. After standardizing quality control metrics, we merged the data from 2174 control and 1772 CTEPH ECs. Dimension reduction of the merged dataset revealed 12 endothelial clusters (Figure). We note disproportionate distribution of control vs CTEPH ECs across clusters, with a majority of clusters biased towards one condition. Between and within clusters, CTEPH ECs displayed greater expression of TGFβ1, a marker of endothelial-mesenchymal transition (EMT), and P-selectin, a marker of endothelial activation. <h3>Conclusion</h3> Significant endothelial heterogeneity exists in both control and CTEPH ECs. However, we find differences in the composition of EC phenotypes between these two groups. We identify marker genes for CTEPH ECs that contribute to our understanding of EC phenotypes and misguided vascular repair in CTEPH.

High-throughput single-cell RNA sequencing.

High-throughput, droplet-based single-cell RNA sequencing (scRNA-Seq) provides unprecedented resolution to examine cell–cell heterogeneity in tissues and organs. Microfluidic channels combine individual cells with uniquely barcoded beads to analyze transcriptomes of thousands of cells in parallel that can reconstruct the biological structure in silico. Individual cells can be traced back through bead/cell-specific barcodes on the oligos, and individual transcript amplification bias is accounted for with unique molecular identifiers (UMIs).