 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 01, 2024 |
| Title |
Spleen, 14 d.p.i., NP-specific B cells, BCR-seq |
| Sample type |
SRA |
| |
|
| Source name |
Spleen
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: NP+B220+
genotype: C57BL6/J
treatment: NP-KLH + Alum + LPS
|
| Treatment protocol |
Seven to eight week old mice were immunized intraperitoneally with 100 μg NP(23)-KLH, NP(25)-KLH or NP(27)-KLH (Biosearch Technologies) mixed with 50% (v/v) imject alum (Thermo Fisher Scientific) and 1 μg LPS (Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Splenic and bone marrow cells (tibia, femur and iliac crest) were isolated from C57BL/6J mice immunized with NP-KLH, alum and LPS on 14, 21 and 35 d.p.i.. Splenocytes and BM cells were washed and prepared as single-cell suspensions in MACS buffer. Enriched CD138+ cells were obtained by using Easysepä Release Mouse Biotin Positive Selection Kit. Cell concentration was adjusted to 100 x 106 cells per mL and incubated with 50 μL of normal rat serum for 5 minutes. Biotinylated anti-CD138 (281.2) antibody (1.0 μg/mL) was added to the cells and incubated for 5 minutes. Next the cells were incubated (5 minutes) with Easysepä Biotin positive selection antibody (50 μL/mL) followed by addition of Easysepä Rapidspheres (100 μL/mL) (5 minutes). MACS buffer was added to the cocktail and an Easysepä magnet was used to remove cells that were not bound to rapidspheres. After three washes, CD138+ cells were obtained by incubating with Easysepä Release Buffer for 5 minutes and placing the cells in a magnet to release them from the Rapidspheres. Purity of enriched CD138+ cells was determined by flow cytometry after labeling a small fraction of the cells with viability dye eFluor 780 (eF780), APC Streptavidin and PE-Dazzle Red 594 B220 (RA3-6B2) for 30 minutes at 4˚C. For the isolation of CD138+ subsets by flow cytometry, enriched CD138+ cells after Rapidspheres release, were labeled with viability dye eFluor 780 (eF780), APC-Streptavidin, PE-Dazzle Red 594 B220 (RA3-6B2), PerCP-Cy5.5 CD44 (IM7) and FITC CD11a (M17/4) for 30 minutes at 4˚C and were sorted as B220+CD138+CD44-CD11a-, B220+CD138+CD44+CD11a+, B220intCD138+CD44+CD11a+ and B220-CD138+CD44+CD11a+ subsets using FACSAria II (BD) with 70 μm nozzle at 4˚C.
Enriched CD138+ splenocytes or flow sorted CD138+ splenic and BM subsets were suspended in 5% FBS and loaded along with oil and beads containing unique barcodes onto the Chromium controller (10x Genomics) to form an emulsion. Single cells contained in oil droplets were then lysed and mRNA was captured by the beads and subjected to reverse transcription and cDNA amplification to generate libraries for sequencing, according to the manufacturer’s protocol, as described previously2. From the cDNA libraries, one aliquot was used for fragmentation and preparation for transcriptome profiling, and another for amplification of VDJ regions using the BCR kit. Samples were then sequenced using a NovaSeq 6000 (Illumina) in which 30,000 and 5,000 reads were generated with the mRNA and BCR libraries respectively.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10X genomics - 5Prime - V1
filtered_contig_annotations.csv
filtered_contig.fasta
Experiment2_D14_SP_BCR
|
| Data processing |
For all scRNA-Seq experiments, UMI counts were calculated using CellRanger (v.3.1.0) and reference genome set to mm10. CellRanger filtered feature sparse matrix counts files from the CD138+ splenocyte sample obtained on 35 d.p.i. (HDF5 format) were supplied for unsupervised analysis using ICGS2 algorithm with default parameters. Cell-type predictions were initially obtained from ICGS2 based on marker enrichment and then further refined based on manual curation with literature-associated markers. Non-B-lineage cells were identified according to their marker genes, and thereby excluded from the downstream analysis. The obtained ICGS2 clusters were filtered to include B cell and plasma cell clusters for downstream UMAP visualization.
For all single-cell experiments, the ICGS generated cell clusters defined for Day 35 cells from Experiment1 were used as the reference sets in the cellHarmony pipeline4. For each scRNA-Seq dataset, the marker genes defined for each ICGS2 cluster from Day 35 sample were used as the reference cluster markers and log-transformed normalized gene expression files were provided as the input for cellHarmony pipeline. The correlation cutoff parameter in cellHarmony was set to 0.7 to include only the cells with stronger matches to the reference cell clusters, thereby excluding low quality cells.
Single-cell VDJ sequencing data were processed using Cellranger vdj (v.3.1.0) with refdata-cellranger-vdj-GRCm 38-alts-ensembl-3.1.0 reference from 10x Genomics. Barcode information obtained from the CellRanger VDJ output and gene expression were used for coupling the BCR and transcriptomes respectively. Transcriptomes were labeled based on ICGS2 clusters obtained on 35 d.p.i. or cellHarmony clusters in the case of other datasets. Cells that did not contain both the BCR and gene expression features were excluded for any downstream analysis. The hypermutation rate and affinity maturation were determined for these cells as described previously2. For clonal analysis, cells were defined based on the following criteria. Clones were identified based on cells that bear identical V(D)J rearrangements in their heavy and light chain loci (kappa or lambda), including sharing of amino acid sequences encoded in VH CDR3 regions. If a cell contained multiple kappa and/or gamma chain rearrangements, then the productively rearranged light chain gene was used for clonal identity. This analysis was performed for 22 NP-specific VH genes and 19 KLH-specific VH genes.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: scBCR-seq
|
| |
|
| Submission date |
Aug 14, 2023 |
| Last update date |
Feb 01, 2024 |
| Contact name |
Harinder Singh |
| E-mail(s) |
harinder@pitt.edu
|
| Phone |
6503034820
|
| Organization name |
University of Pittsburgh
|
| Department |
Center for Systems Immunology and Department of Immunology
|
| Lab |
Dr Harinder Singh
|
| Street address |
5051 Centre Avenue
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE240813 |
Temporal dynamics and genomic programming of plasma cell fates |
|
| Relations |
| BioSample |
SAMN36984625 |
| SRA |
SRX21362015 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7709783_Experiment2_D14_SP_BCR.tar.gz |
1.9 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
