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RNA sequencing analysis of gene expresssion profiles in Cic-null MZB cells

ABSTRACT: Purpose: The goal of this study is to compare and examine the transcriptional profiles in Cd19-Cre (control) versus Cicf/f;Cd19-Cre (Cic-null) MZB cells,by mRNA sequencing. Methods: MZB cells (CD19+CD93-CD21+CD23-) were sorted by a MoFlo-Astrios (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. . A library for mRNA sequencing was prepared using the Truseq Stranded mRNA/Total Library Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed with Novaseq 6000 (Illumina). Results: Tophat (v2.0.13) was used to map reads for each sample to the mm10 RefSeq reference genome. The aligned results were then added to Cuffdiff (v2.2.0) to identify the differentially expressed genes (DEGs). In total, 775 genes were differentially expressed in Cic-null MZB cells (505 upregulated and 270 downregulated) compared to control cells (Log2 fold-change > 0.5 and P < 0.01). Conclusions: Our study presents the first comparative gene expression analysis of MZB cells from control and B cell-specific Cic-deficient mice. We concluded that CIC deficiency attenuates MZB cell development through downregulation of the NOTCH signaling pathway. The data reported here also provide references for the gene expression profiles of murine MZB cells through TPM.

ORGANISM(S): Mus musculus

PROVIDER: GSE264654 | GEO | 2024/04/29

REPOSITORIES: GEO

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BCR repertoire analysis of wild-type and Cic-null B-1a cells

Project description:Purpose: B-1a cells have a distinct BCR repertoire compared with that of B-2 cells. To examine whether CIC loss affects the BCR repertoire in B-1a cells, we analyzed mRNA sequences of immunoglobulin heavy (Igh) and light (Igk and Igl) chain genes in B-1a cells from 12-week-old control and Cicf/f;Cd19-Cre mice. Methods: Peritoneal cavity B-1a cells (IgM+, CD19+, CD5+, CD43+) were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. Long Read iR-Profile Reagent System (iRepertoire) was used to generate NGS libraries covering BCR chains including Igh, Igk, and Igl. Briefly, nested inside and outside primers selectively amplified all V- and C- regions and incorporated communal adaptors. Following clean up, only target amplicons, which contain 5’ and 3’ communal adaptors, were exponentially amplified. Amplified libraries were multiplexed for sequencing on the Illumina Miseq platform. Sequence reads were de-multiplexed according to the barcode sequences. Results: Trimmed reads were mapped to germline V, D and J reference sequences downloaded from the IMGT database. IgH diversity and the usage of variable (V) segments in heavy (Ighv) chain and light (Igkv and Iglv) chain genes were comparable between control and Cic-null B-1a cells. Analysis of non-templated (N)-nucleotide addition at V(D)J junctions revealed that Cic-null B-1a cells have a higher proportion of zero to two N-nucleotides-containing-BCRs than control cells. Conclusions: Our study presents the first comparative BCR repertoire analysis of wild-type and Cic-null B-1a cells. We concluded that CIC deficiency does not dramatically alter the BCR repertoire in B-1a cells.

2021-12-16 | GSE164147 | GEO

RNA Sequencing Analysis of Gene Expression Profiles in Cic-null B-1a and Follicular B (Fo B) cells

Project description:Purpose: The goal of this study is to compare and examine the transcriptional profiles in control versus Cic-deficient B cells, specifically splenic B-1a and Fo B cells, by mRNA sequencing. Methods: Splenic B-1a (IgM+, CD19+, CD5+, CD43+) and Fo B (B220+, CD93-, CD23+, CD21lo-mid) cells were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. The DNA library for mRNA sequencing was generated using SMART-seq v4 Ultra Input RNA Kit (Clontech, for B-1a cells) and TruSeq RNA Sample Prep Kit v2 (Illumina, for Fo B cells). DNA libraries were multiplexed and pooled for sequencing on the Illumina Hiseq 4000 platform. Results: Trimmed reads were mapped to mouse reference genome (mm10 RefSeq) with HISAT2, a splice-aware aligner, and transcripts were assembled by StringTie with aligned reads, for known transcripts, novel transcripts, and alternatively spliced transcripts. A total of 712 genes were differentially expressed in Cic-null B-1a cells (325 upregulated and 387 downregulated), while 357 genes were differentially expressed in Cic-deficient Fo B cells (180 upregulated and 178 downregulated) when compared with their respective control cells (fold change >1.2 and P < 0.05). Several known CIC target genes, including Etv1, Etv4, Etv5, Spry4, Spred1, Spred2, Dusp4, Dusp6, and Per2, were included in the list of the upregulated differentially expressed genes (DEGs) in either Cic-null B-1a or Fo B cells or both, validating the reliability of RNA sequencing results. Conclusions: Our study presents the first comparative gene expression analysis of B-1a and Fo B cells from control and B cell-specific Cic-deficient mice. We concluded that CIC deficiency promotes B-1a cell development through enhancing the BCR signaling pathway. The data reported here also provide references for the gene expression profiles of murine B-1a and Fo B cells.

2021-12-16 | GSE163455 | GEO

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Project description:Purpose : The goal of this study is to compare the gene expression profiles between wild-type (WT) and CIC-deficient DP thymocytes to understand the molecular mechanisms of CIC regulation of T cell development, especially in DP thymocytes. Methods : DP thymocytes (lin-CD4+CD8+TCRlo (Lineage : CD11b, CD11c, CD19, NK1.1, Gr-1, γδTCR, and TER119)) from thymus were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using RiboEX (GeneAll) according to the manufacturer's instructions. The library for mRNA sequencing was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) and sequencing was performed with NovaSeq 6000 (Illumina). Results : Trimmed reads were mapped to the mouse reference genome (mm10 RefSeq) with HISAT2, and the transcripts were assembled using StringTie. A total of 482 differentially expressed genes (DEGs; fold change > 2 and P-value < 0.05) were generated using edgeR. The 263 upregulated genes included known CIC target genes such as Etv1, Etv4, Etv5, Spry4, Spred1, Dusp4, and Dusp6. Conclusions : Our study represents the first comparative analysis of DP thymocytes from WT and hematopoietic lineage cell-specific Cic deficient mice. We concluded that CIC deficiency leads to impaired positive and negative selection, and TCR signaling in DP cells. The attenuation of TCR signaling is caused by derepression of CIC target genes involved in dephosphorylation of ERK.

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SLAMF5 blocking of sorted Bregs CD19+IL10+ (GFP+)

Project description:Purpose: Our results show that in the absence of SLAMF5 ( by KO or blocking ab), there is an accumulation of Bregs (CD19+IL10+) and a specific increase of their survival. Here, using RNAseq, we wished to identify genes that take place in the regulation of Breg by SLAMF5 blocking. Method: Total RNA was extracted from 3x10e5 sorted CD19+GFP+cells from 2-3 Vert-x mice (induced with EAE) incubated with SLAMF5 blocking Ab or control IgG using the Direct-zol RNA kit (ZymoResearch) according to the manufacturer’s instructions. RNA-seq was performed using the MARS-seq protocol. Showing here 2 biological replicates of each group.

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