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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 15, 2024 |
| Title |
+/+_cre Rep1 |
| Sample type |
SRA |
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| Source name |
Spleen cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen cells
cell type: Germinal center B cells
genotype: +/+ cre
treatment: FACS sorted
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the EZ-10 DNAaway RNA kit (Bio Basic), according to the manufacturer’s protocol, and quantified with NanoDrop (ThermoFisher Scientific). cDNA was prepared using the Moloney murine leukemia virus (MMLV) reverse-transcription kit and quantitative PCRs run on a StepOnePlus instrument with PowerSYBR master mix (all from ThermoFisher Scientific). Primers were purchased from Integrated DNA Technologies.
RNA yields and quality were assessed using Bioanalyzer (Agilent). rRNA depletion and library preparation were performed using the KAPA RNA HyperPrep Kit with RiboErase (Roche).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Total B cells were isolated from mouse spleens via magnetic enrichment using with the EasySep Mouse CD19 Positive Selection Kit II (Stem Cell Technologies). For the FACS-sorting of B cell subsets, mouse spleens were mechanically dissociated in PBS with 0.1% BSA and 2mM EDTA, passed through 40 μm cell-strainers, and subjected to red blood cell lysis in ACK buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA). Cells were stained with the following antibodies: APC anti-CD267/TACI (ebio8F10-3, eBioscience); Alexa Fluor 488 anti-GL7 (GL7, Biolegend); Brilliant Violet 421 anti-CD95/Fas (Jo2, BD Biosciences) and anti-CD138 (281-2, Biolegend); Brilliant Violet 650 anti-CD45R/B220 (RA3-6B2, BioLegend); Biotin anti-IgG1 (polyclonal, 1070-08, Southern Biotech); PE-Cy7 anti-CD19 (6D5, BioLegend); PerCP-Cy5.5 antiCD4 (RM4-4, Biolegend), anti-CD8a (53-6.7, Biolegend), anti-CD11b (M1/70, eBioscience), anti-NK1.1 (PK136, Biolegend), and anti-TER119 (Ly-76, Biolegend). Brilliant Violet 785 Streptavidin (Biolegend) was used to detect biotin-conjugated antibodies. Viability Dye 7-AAD (Biolegend) or eFluor® 506 (eBioscience) was used to discriminate dead GC B cells or dead plasma cells, respectively. Sorting was performed on the FACSAria (BD Biosciences).
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| Data processing |
The quality of the sequencing reads was confirmed using the FastQC tool (Babraham Bioinformatics), and low-quality bases were trimmed from the read extremities using Trimmomatic v.0.33.The reads were then mapped to the mouse UCSC mm9 reference genome assembly using Hisat2 v2.2.1. Gene expression was quantified by counting the number of uniquely mapped reads with featureCounts using default parameters.
We retained genes that had an expression level of at least 5 counts per 106 reads (CPM) in 4 samples for GC cells and 1 sample for plasma cells, and performed quantile normalization with the preprocessCore package to remove batch effects. TMM normalization and differential gene expression analyses were conducted using the edgeR Bioconductor package. Dimension reduction analysis was performed using the Principal Component and Partial Least Squares regression method.
Pairwise comparisons were performed between the genotypes or between the treatments, and genes with changes in expression ≥ |2.0| fold and Benjamini-Hochberg adjusted p values ≤ 0.05 were considered significant.
For data visualization in Integrative Genomics Viewer (IGV) bigwig files were generated using a succession of genomeCoverageBed and wigToBigWig tools and scaled per 10x106 exon-mapped reads. Gene ontology (GO) enrichment analyses on differentially expressed gene clusters were performed with DAVID Bioinformatics Resources 6.8, and Gene Set Enrichment Analysis (GSEA) was performed with GSEA tool v4.3.2 using MSigDB v2022.1 with default configuration and permutation within gene sets.
Assembly: mm9
Supplementary files format and content: tab-delimited text files include feature counts for each sample, where rRNA have been excluded.
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| Submission date |
Oct 04, 2023 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Anastasia Nijnik |
| E-mail(s) |
anastasia.nijnik@mcgill.ca
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| Phone |
514-398-5567
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| Organization name |
McGill University
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| Department |
Physiology
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| Lab |
Nijnik Lab
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| Street address |
Rm 368 - 3649 Promenade Sir William Osler
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 0B1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE244623 |
B-cell Intrinsic Regulation of Antibody Mediated Immunity by Histone H2A Deubiquitinase BAP1 [RNA-seq] |
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| Relations |
| BioSample |
SAMN37682814 |
| SRA |
SRX21987918 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7822296_C1_rmOverRep_merged_featurecounts_no_rRNA.txt.gz |
199.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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