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| Status |
Public on Mar 29, 2021 |
| Title |
ImmatureB_KO_3 |
| Sample type |
SRA |
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| Source name |
Immature B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 10w 6d
gender: m
tissue: Bone marrow
cell type: Immature B cells (Bone marrow cells sorted by gates B220+CD3-CD11b-NK1.1-CD11c-TER119-Gr1-CD19+IgM+IgD-)
genotype: Bap1 fl/fl mb1-Cre
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| Growth protocol |
The mice were maintained under specific pathogen-free conditions. Test and control groups were bred as littermates and co-housed together.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Bone marrow was flushed in PBS supplemented with 0.1% BSA and 2mM EDTA, filtered through 40μm cell-strainers, and subjected to red blood cell lysis in ACK buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA). The samples were stained with: biotin-conjugated antibodies against lineage markers [anti-CD3ε (145-2C11, BioLegend), anti-CD11b (M1/70, BioLegend), anti-CD11c (N418, BioLegend), anti-Ly-6G/Ly-6C (Gr-1) (RB6-8C5, BioLegend), anti-NK-1.1 (PK136, BioLegend), and anti-TER-119 (TER-119, BioLegend)], followed by APC-eFluor 780-conjugated Streptavidin (eBioscience), Alexa Fluor 488-conjugated anti-IgD (11-26c.2a, BioLegend), APC-conjugated anti-CD43 (S7, BD Biosciences), PE-conjugated anti-IgM (II/41, eBioscience), PE-Cy7-conjugated anti-CD19 (6D5, BioLegend), and PerCP-Cy5.5-conjugated anti-CD45R/B220 (RA3-6B2, BioLegend) antibodies. DAPI was added immediately before sorting for dead cell exclusion. Cell sorting was performed on FACSAria and analyzed with FACS Diva software (BD Biosciences). The cells were sorted using FACS into pre-B and immature B cells, gating on B220+CD3-CD11b-NK1.1-CD11c-TER119-Gr1-, and IgM-IgD-CD19+CD43- for pre-B cells, and CD19+IgM+IgD- for immature B cells.
RNA was isolated from the FACS-sorted cells using the MagMAX total RNA kit (Ambion, Life Technologies), and quality assessed using Bioanalyzer RNA Pico chips (Agilent). rRNA depletion and library preparation were performed using the SMARTer Stranded RNA-Seq kit (Takara Clontech). The libraries were sequenced on an Illumina HiSeq 4000 sequencer in paired-end 50bp configuration. Cells from multiple mice were pooled to achieve yields of >1.7ng of RNA per sample, and 3-4 independent samples were analyzed per genotype for each cell-type.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
The high quality of sequence reads was confirmed using FastQC tool.
low-quality bases were trimmed from read extremities using Trimmomatic v.0.33.
Trimmed reads were then mapped to the mouse UCSC mm9 reference assembly using TopHat v2.0.9 in conjunction with Bowtie 1.0.0 algorithms.
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 minimum 5 counts per million reads (CPM) in at least 3 of the samples, 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 (R version 3.4.2).
Pairwise comparisons were performed between samples across different mouse genotypes and genes with changes in expression ≥ |1.5| folds and Benjamini-Hochberg adjusted p values ≤ 0.01 were considered significant.
Genome_build: mm9
Supplementary_files_format_and_content: Matrix table with CPM values after quantile normalization
Supplementary_files_format_and_content: Tab-delimited text with raw gene counts for every gene in each sample as generated by featureCounts (gene annotation: UCSC mm9)
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| Submission date |
Nov 24, 2020 |
| Last update date |
Mar 29, 2021 |
| 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 |
GPL21103 |
| Series (2) |
| GSE162084 |
Regulation of B lymphocyte development by histone H2A deubiquitinase BAP1 (Bap1KO_RNA-Seq) |
| GSE162085 |
Regulation of B lymphocyte development by histone H2A deubiquitinase BAP1 |
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| Relations |
| BioSample |
SAMN16881979 |
| SRA |
SRX9567569 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4932869_ImmatureB_KO_3_featurecounts_no_rRNA.txt.gz |
158.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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