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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 28, 2023 |
| Title |
N/IsdB |
| Sample type |
SRA |
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| Source name |
N/IsdB
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen and kidneys
isdb vaccinated: vaccinated with IsdB
lac infection: Naïve mice
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| Treatment protocol |
Mice were immunized i.p. three times with IsdB (75μg, 50μg and 50μg) plus aluminum hydroxide (alum, InvivoGen) (450 μg per dose) or with aluminum hydroxide alone at 7-day intervals. Mouse sera were screened for reactivity to IsdB by ELISA. IsdB-specific antibodies were purified from human or mouse sera using immobilized IsdB agarose columns (NHS-activated agarose, ThermoFisher Scientific).
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| Growth protocol |
S. aureus Becker and S. aureus Becker IsdB/HarA deletion mutant were gifts from Dr. Secore (Merck). Overnight S. aureus cultures were diluted 1:200 in Todd Hewitt broth (THB) and grown to an optical density of 0.8. Unless otherwise stated, 6-8 weeks old female mice were administered 2x107 LAC (USA 300) i.p. for each S. aureus challenge. Spleen and kidneys were harvested 24 hr after the last infection, homogenized in phosphate-buffered saline (PBS) and plated on THB agar plates for CFU enumeration. Other S. aureus inoculums were: 5x107 LAC i.p. for the LD90 infection, 2x107 SA113 i.p., 4x107 Newman or Becker (WT or IsdB/HarA mutant) i.p., 4x107 LAC s.c., and 1x106 LAC i.v.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Opsonophagocytosis killing assay (OPK) was performed. Mouse neutrophils were isolated from bone marrow by MojoSort™ Mouse Neutrophil Isolation Kit (BioLegend). Overnight culture of S. aureus LAC was sub-cultured 1:200 in THB and grown to an optical density of 0.6. S. aureus was washed, resuspended in PBS, and incubated with mouse sera at 37°C for 20 min, then added to 105 mouse neutrophils a multiplicity of infection (MOI) of 1:0.5 in the presence of 2% normal mouse serum. Following incubation at 37°C for 1 hr with agitation at 200 rpm, samples were plated on THB agar plates for CFU enumeration.
Splenocytes from IsdB immunized or LAC-experienced mice were incubated with phycoerythrin (PE)–labeled IsdB and allophycocyanin-conjugated anti-mouse CD45R/B220. IsdB+ B220+ cells were sorted by FACSAria II (BD) and subjected to single-cell preparation by using a Single Cell 5’ Library and Gel Bead kit and Chromium Single Cell A Chip kit, the cell suspension was loaded onto a Chromium single cell controller to generate single-cell gel beads in the emulsion (GEMs) according to the manufacturer’s protocol (10X Genomics). scRNA-seq libraries were constructed using a Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell following instructions provided by the manufacturer (10X Genomics). The libraries were sequenced using an Illumina Novaseq6000 sequencer with a paired-end 150-bp (PE150) reading strategy (performed by Institute for Genomic Medicine, UCSD).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The BCR sequence data were processed using the Immcantation toolbox (v4.0.0) with default parameter values. Initial germline V(D)J gene annotation was performed using IgBLAST with IMGT germline sequence databases.The IgBLAST database was further used to assign V(D)J gene annotations to the BCR FASTA files for each sample using the Change-O package. The derived matrix contains sequence alignment information for each sample with both light and heavy chain sequences, and individualized genotypes were inferred using the TIgGER package and used to finalize V(D)J annotations.
The clonal groups were identified by the R package – scRepertoire (11) based on paired heavy and light chains. To determine clonal groups, we first used the filtered contig annotation obtained from the results performed using the Cell Ranger Single-Cell Software Suites (http://software.10xgenomics.com/single-cell/overview/welcome). Then, for the cells with high quality paired heavy and light chains were sequenced, clones were assigned based on strict definition of clonotype using the CTstrict() function that considers clonally related two sequences with identical V gene usage and > 85% normalized Levenshtein distance of the nucleotide sequence. The clonotype changes between samples were visualized by the compareClonotypes() function with the clones called by amino acid sequence of the CDR3 region.
Supplementary_files_format_and_content: clonotypes.csv, consensus.fasta, and filtered_contig_annotations.csv.gz
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| Submission date |
Jan 12, 2022 |
| Last update date |
Jan 28, 2023 |
| Contact name |
Nathan E. Lewis |
| E-mail(s) |
n4lewis@ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
Pediatrics
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| Street address |
9500 Gilman Dr.
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92123 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE193543 |
Non-protective immune imprint underlies failure of S. aureus IsdB vaccine |
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| Relations |
| BioSample |
SAMN24899240 |
| SRA |
SRX13753845 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5814002_5_NT-IsdB-B.clonotypes.csv.gz |
59.3 Kb |
(ftp)(http) |
CSV |
| GSM5814002_5_NT-IsdB-B.consensus.fasta.gz |
224.3 Kb |
(ftp)(http) |
FASTA |
| GSM5814002_5_NT-IsdB-B.filtered_contig_annotations.csv.gz |
429.4 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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