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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 27, 2023 |
| Title |
Splenic CD19+ Ig transcript library, WT C57BL/6J sample 3 |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: CD19+
strain background: C57BL/6J
genotype: WT
Sex: Female
age: 10.5 weeks
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| Extracted molecule |
total RNA |
| Extraction protocol |
CD19+ splenocytes were isolated 10.5-week-old 3 WT and 3 Mtv8KO C57BL/6J female mice. Red blood cell lysed splenocytes were labeled with microbeads conjugated to monoclonal anti-mouse CD19 antibodies (isotype: rat IgG2a) (Miltenyi Biotec, Bergisch Gladbach, Germany) and positively sorted as detailed by the manufacturer. RNA was isolated from isolated CD19+ splenocytes using guanidine thiocyanate extraction and CsCl gradient centrifugation.
Immunoglobulin libraries were generated from 1 mg of CD19+ splenocyte RNA using the NEBNext Immune Sequencing Kit (New England Biolabs, Ipswich, Massachusetts, USA) according to manufacturer’s instructions, specifically enriching for B cell receptor chains during the first PCR step.
Six libraries were pooled in equimolar amounts and were sequenced by paired-end 300 bp sequencing on an Illumina MiSeq by The University of Chicago Genomics Facility.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
Immunoglobulin libraries were generated from 1 mg of CD19+ splenocyte RNA using the NEBNext Immune Sequencing Kit, specifically enriching for B cell receptor chains during the first PCR step
Mtv8_Ig_Repertoire_Summary.xlsx
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| Data processing |
Preprocessing of BCR sequences was performed using the open-source workflow pRESTO NEBNext Immune Sequencing Kit Workflow (v3.2.0) on Galaxy
Reads with a Phred quality score <20 were removed for quality control. Reads that did not match to the constant region primer (maximum error rate 0.2) were removed. Reads that did not match to the template switch sequence (maximum error rate 0.5) were removed. The first 17 bp following the template switch site were a unique molecular identifier (UMI) on each read. Sequences with identical UMIs were collapsed into consensus sequences with sequences found in less than 60% of reads removed. Positions with more than 50% gap sequences were removed. Mate-pairs were assembled with a minimum of 8 bp overlap (maximum error rate of 0.3). Assembled reads were assigned isotype-constant region identities based on local alignment of the 3’ ends of the reads (maximum error rate of 0.3).
V, (D), and J gene segments were assigned using MiGMAP mapper (Galaxy Version 1.0.3+galaxy2).
Subsequent analyses were done using R Studio. Statistically significance was calculated using unpaired Welch’s t test with Bonferroni correction.
Assembly: mm10
Supplementary files format and content: Final Ig repertoire summary and raw counts: Mtv8_Ig_Repertoire_Summary.xlsx
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| Submission date |
Nov 21, 2023 |
| Last update date |
Nov 27, 2023 |
| Contact name |
Helen A Beilinson |
| E-mail(s) |
beilinsonh@uchicago.edu
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| Organization name |
University of Chicago
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| Department |
Microbiology
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| Street address |
924 E 57TH ST
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE248410 |
The endogenous retrovirus Mtv8 which resides within the V kappa locus does not influence the mouse immunoglobulin repertoire |
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| Relations |
| BioSample |
SAMN38351342 |
| SRA |
SRX22600075 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7913096_HB3_Galaxy328-_MiGMAP_on_data_266.tabular.txt.gz |
25.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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