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Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

ABSTRACT: Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

PROVIDER: PRJNA811523 | ENA |

REPOSITORIES: ENA

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Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

Project description:Purpose:Flow cytometric analysis showed that after 48h of culture with T cell- and Ag-derived stimuli, GCBC alter their TF expression and begin to differentiate. However, only a subset of cells undergoes this process, for reasons that are unclear. To better define the nature of this heterogeneity, and to further define the differences between IL-21R/CD40 vs BCR/CD40 signals, we performed single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation. Methods: we performed scRNA-seq using 10X Genomics Chromium system . Results: Single cell analysis demonstrated surprisingly little overlap between the clusters derived from IL-21/⍺-CD40 stimulation and those derived from BCR/⍺-CD40 ligation, thus establishing at molecular and cellular detail how distinct these two types of selection signals are for GCBC

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RNA-seq analysis of GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these.

Project description:Purpose: To gain insight into both similarities and differences in the gene expression programs induced by IL-21R, BCR and CD40 signals, we performed RNA-seq on both GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these. Methods : Purified GCBC from d14 NP-CGG immunized MEG mice and NBC from naïve MEG mice were warmed at 37C for 30 minutes and then stimulated with 10ng/mL IL-21, 20μg/ml ⍺-IgM or 2.5μg/mL ⍺-CD40 (prepared as tetramer) alone or combined IL21/⍺-CD40 or ⍺-IgM/⍺-CD40 for 2 and 4 hours. After stimulation, cells were processed for RNA isolation using QIAshredder columns (Qiagen) and the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. Samples were sequenced using Illumina NextSeq 500 with 75 bp paired-end reads and aligned to the mm10 genome using the STAR aligner (Dobin et al., 2013). Gene-level counts were determined using featureCounts (Liao et al., 2014), and raw counts were quantile normalized to each other for differential expression using the voom method (Law et al., 2014) in the Limma R package (Ritchie et al., 2015). For normalization of the datasets, the Quantile method was used. Results: Like BCR and CD40 signals, IL-21R plus CD40 signals also synergize to induce c-Myc in GCBC. However, IL-21R plus CD40 stimulation differentially affects GCBC fate compared to BCR plus CD40 ligation—engaging unique molecular mechanisms

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