D8429535-2996-4469-a85f-cb12bda3649d - samples
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ABSTRACT:
PROVIDER: EGAD00001009831 | EGA |
REPOSITORIES: EGA
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Project description:Sorted single CD8+T cells expressing CD14 from human liver for SMARTSeq2.
Livers processed: Kucykowicz et al STAR Prot 2022:pubmed.ncbi.nlm.nih.gov/35516846/
Published: Pallett et al Nature 2022
Tissue CD14+CD8+T-cells are reprogrammed by myeloid cells and modulated by LPS
The liver is bathed in bacterial products including LPS transported from the intestinal portal vasculature, but maintains a state of tolerance exploited by persistent pathogens and tumours1,2,3,4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+T-cells compartmentalised in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins; CD14+CD8+T-cells preferentially accumulate within the donor pool in liver allografts, amongst hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+T-cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10/IL-2 production directly ex vivo, and an enhanced capacity to rapidly produce antiviral/anti-tumour cytokines upon TCR engagement. This CD14+CD8+T-cell profile can be recapitulated by acquisition of membrane proteins, including the LPS receptor complex, from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T-cells in vitro. CD14+CD8+T-cells express integrins and chemokine receptors favouring interactions with the local stroma, which can promote their induction through CXCL12. LPS can also increase the frequency of CD14+CD8+T-cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+T-cells with immunomodulatory capacity.
For the analysis of the single cell transcriptome two intrahepatic perfusate samples were isolated and frozen at -80 'C for storage prior to transportation to Newcastle, UK. Once thawed in pre-warmed HBSS containing 0.001% DNaseI, intrahepatic leukocytes were sorted by FACS to index classical mononuclear phagocytes (MNP) defined as Singlets, CD3-CD56-CD19-CD14hiHLA-DR+ or CD14± CD8+T-cells as follows: Singlets, CD3+CD56-CD19-CD4-CD8+Vï¡7.2- and split in to CD14± based on an isotype control. Single cells were sorted into 96-well skirted LoBind plates (Eppendorf) containing 5 µL lysis buffer (TCL buffer [Qiagen], supplemented with 1% b-mercaptoethanol [Sigma Aldrich; Merck]) on a BD Bioscience FACSFusion. The sorted plates were subsequently immediately sealed, centrifuged, and placed on dry ice to flash-freeze the cell lysate and stored at -80 'C.
A modified SMART-seq2 protocol57 was performed on the single flow cytometry sorted-cells as previously described58. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit (Illumina). Each library was sequenced to achieve a minimum depth of 1-2 million raw reads per cell using an Illumina HiSeq 4000 using v. 4 SBS chemistry to generate 75-bp paired-end reads.
| EGAS00001006885 | EGA
Project description:similar methods as in Gomez-Picos et al. (2022) and Nguyen et al. (2023)
2023-12-18 | GSE249932 | GEO
Project description:Purpose: The aim of this study is to determine the absolute and relative expression levels of mRNA transcripts across multiple flow cytometrically sorted epithelial cell types including freshly isolated CD24+CD29hi mammary stem cell-enriched basal cells (MaSC/basal), CD24+CD29loCD61+ luminal progenitor-enriched (LP) and the CD24+CD29loCD61- mature luminal-enriched (ML) cell populations. Additionally, a comparison between these primary cell types and cultured MaSC/Basal-derived mammosphere cells (mammosphere) and the CommaD-βGeo (CommaDβ) cell line was performed. Methods: Total RNA was extracted and purified from sorted luminal or basal populations from the mammary glands of female virgin 8- to 10-week-old FVB/N mice (3 independent samples per population), MaSC/Basal cells cultured for 1 week under mammosphere conditions and CommaDβ cells grown under maintenance conditions (Deugnier et al. 2006). Total RNA (100 ng) was used to generate sequencing libraries for whole transcriptome analysis following Illumina’s TruSeq RNA v2 sample preparation protocol. Completed libraries were sequenced on HiSeq 2000 with TruSeq SBS Kit v3- HS reagents (Illumina) as 100 bp single-end reads at the Australian Genome Research Facility (AGRF), Melbourne. Approximately 30 million 100 bp single-end reads were obtained for each sample. Reads were aligned to the mouse reference genome mm10 and mapped to known genomic features at the gene level using the Rsubread package (version 1.14.1) (Liao et al. 2013). Single reads were then summarized into gene-level counts using FeatureCounts (Liao et al. 2014).
2015-03-15 | GSE63310 | GEO
Project description:Purpose: The aim of this study is to identify genes that are under the transcriptional control of the epigenetic modifier Smchd1 in mouse lymphoma cell lines. Methods: Total RNA was extracted using QIAGEN RNeasy Minikit from sorted lymphoma cell lines derived from mice either wild-type or null for Smchd1. 1µg total RNA was used to generate sequencing libraries for whole transcriptome analysis with Illumina’s TruSeq RNA Sample Preparation Kit v2 as per standard protocols. Libraries were sequenced on HiSeq 2000 with Illumina TruSeq SBS Kit v3-HS reagents as either 100 bp single-end or paired-end reads at the Australian Genome Research Facility (AGRF), Melbourne. Reads were aligned to the mouse reference genome mm10 and mapped to known genomic features at the gene level using the Rsubread package (version 1.10.5) (Liao et al. 2013). Mapped reads were then summarized into gene-level counts using FeatureCounts (Liao et al. 2014).
2015-03-13 | GSE64099 | GEO
Project description:VSMCs expressing SCA1 have increased proliferative capacity (Dobnikar et al, 2018; Worssam et al, 2022; Pan et al, 2020). We therefore, mapped chromatin accessibility changes using bulk ATAC-seq for SCA1+ and SCA1- lineage traced VSMCs.
2024-04-09 | GSE246646 | GEO
Project description:Preparation of sequencing libraries and sequencing analysis using Illumina HiSeq 3000 platform (50 bp single-read module) were conducted at the Genomics Core of UT Health San Antonio. Sequencing reads were preprocessed by Trim Sequences tools in CLC Genomics workbench. Remaining reads were then aligned to mouse GRCm38(mm10) reference genome using RNA-Seq Analysis module in CLC Genomics workbench. To determine differentially expressed genes, exact test were performed after filtering lowly expressed gene (counts per million < 1 in more than three samples across the dataset) using edgeR package (Robinson, McCarthy et al. 2010). Gene Ontology (GO) analysis were performed using DAVID (Dennis, Sherman et al. 2003) and then visualized by GOplot (Walter, Sanchez-Cabo et al. 2015).
2020-04-16 | GSE133140 | GEO
Project description:Purpose: The goal of this study is to identify differentially expressed genes in SETDB2 deficient mouse primary hepatocytes (MPH) treated with dexamethasone when compared to control MPH Methods: Total RNA from MPH treated with either shControl or shSETDB2 adenovirus (pool of 3 mice per group) was isolated by the TRIzol method (Life Technologies) according to manufacturer’s instructions. Total RNA was DNase-treated and RNA integrity was evaluated by Biolanalyzer (Agilent). RNA sequencing was performed by the SBP Genomics Core Facility in Lake Nona (Orlando, FL) using the Illumina HiSeq platform and the following parameters: paired-end read length: 100 BP; number of output reads: 300m reads. The Tophat+Cufflinks pipeline was used for the RNA-seq analysis as described previously (Haas et al., 2013; Trapnell et al., 2012). Differentially expressed genes in shSETDB2 vs. shControl MPH were determined using a 2-fold cutoff, p-value of less than 0.05, and FPKM (fragments per kilobase of transcript per million mapped reads) cutoff of 1. The shSETDB2 and shControl RNA-seq reads were mapped to the mm10 genome using Tophat (Trapnell et al., 2009). The mapping results were further analyzed by Cufflinks, which outputs the final differential gene list.
2016-08-28 | GSE85278 | GEO
Project description:RNASeq was performed on mRNA obtained from OMP expressing and TRPC2 expressing olfactory sensory neurons from 48hpf zebrafish embryos. Single-cell suspensions of 48hpf olfactory epithelia were FAC-sorted to obtain RFP expressing or venus expressing cells (RFP sample1: 3500 cells, RFP sample2: 5500 cells. Venus sample1: 676 cells, venus sample2:201 cells. RNA was extracted using QIAGEN RNEasy kit and cDNA was synthesized using a custom oligo-dT primer containing T7 promoter sequence courtesy of Jim Eberwine, University of Pennsylvanina. In vitro RNA amplification was carried out through one round for OMP:RFP neurons and through two rounds for TRPC2:venus neurons as per the protocol in Morris et al, 2011. Adapter-tagged libraries were synthesized using Illumina TruSeq v2.0 and deep-sequenced on HiSeq2500 to obtain ~100million reads per sample.
2018-03-03 | GSE86023 | GEO
Project description:The immune system of SPF mice is dominated by naïve phenotype immune cells, especially within the T cell compartment, and resembles that of neonatal humans (Beura et al., 2016; Reese et al., 2016). Sequential infection of SPF-housed laboratory mice with common experimental pathogens, such as MHV, MCMV, Listeria monocytogenes, LCMV, and influenza A virus (Berton et al., 2022; Reese et al., 2016), or cohousing (CoH) SPF mice with pet store mice carrying multiple pathogenic and commensal bacteria, viruses, and/or fungi (Beura et al., 2016) induces immune system alterations and maturations that more closely resemble the adult human immune system. Such microbial exposure drastically alters the composition and function of the immune system of laboratory mice, which can significantly influence the overall outcome (and survival) to subsequent infection. Here we report CD11b+ CD115+ monocytes sorted from female specific pathogen-free (SPF) C57BL/6N (B6) mice co-housed with petstore mice for 60 days are transcriptionally different than SPF CD11b+ CD115+ monocytes
2023-10-26 | GSE237883 | GEO