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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 24, 2020 |
| Title |
Spleen 2 |
| Sample type |
SRA |
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| Source name |
IgG+ CD38+ CD138- GL7- CD19+ cells spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: spleen
mouseid: 2
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| Treatment protocol |
3x NP-CGG immunized C57BL/6 mice
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single-cell suspensions from BM of 3x NP-CGG immunized C57BL/6 mice were prepared and CD19+ cells were enriched by magnetic cell sorting using anti-CD19 microbeads (Miltenyi Biotech). Ex vivo IgG1+/IgG2b+CD19+CD38+GL7-CD138-IgM-IgD- memory B cells were isolated by FACS (Influx cell sorter (BD Bioscience)) and applied to the 10X Genomics platform using the Single Cell 5’ Library & Gel Bead Kit (10x Genomics) following the manufacturer’s instructions.
The amplified cDNA was used for simultaneous 5’ gene expression (GEX) and murine BCR library preparation. BCR transcripts were additionally amplified by Chromium Single Cell V(D)J Enrichment Kit for murine B cells (10x Genomics). Upon adapter ligation and index PCR, the quality of the obtained cDNA library was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using a High Output v2 Kit (150 cycles) with the recommended sequencing conditions (read1: 26nt, read2: 98nt, index1: 8 nt, index2: n.a.) for the transcriptome and Mid Output v2 Kit (300 cycles) for BCR repertoire analysis (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Spleen 2
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| Data processing |
The Cell Ranger Single Cell Software Suite 2.1.1 was used to perform sample demultiplexing, barcode processing, and single cell 3’ gene counting for transcriptome wirh refdata-cellranger-mm10-1.2.0 ar reference and expected-cells number of 3000 for each sample.
For immune profiling Cell Ranger Single Cell Software Suite 3.0.2 was used for demultiplexing, barcode processing and assembly of the BCR sequences, using the vdj command and refdata-cellranger-vdj_GRCm38_alts_ensembl-mouse-2.2.0 as reference.
Genome_build: mm10
Supplementary_files_format_and_content: Barcoded BAM, with Chromium cellular and molecular barcode information for each read, stored as TAG fields; h5: aggregated gene count matrix in hdf5 format. fastq: high-confidence contig sequences of immune receptors annotated with the corresponding cellular barcode and contig-id; csv: results from the signle cell immune profiling comprising including the receptor type, VDJ-Genes annotation, CDR3 nucleotide and amino acid sequense as well as the count for the corresponding cellular barcode and contig.
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| Submission date |
Nov 01, 2019 |
| Last update date |
Apr 25, 2020 |
| Contact name |
Pawel Durek |
| E-mail(s) |
pawel.durek@drfz.de
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| Organization name |
Deutsches Rheuma-Forschungszentrum
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| Street address |
Charitéplatz 1
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| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE139836 |
Single-Cell transcriptomes and immune profiling of IgG+ murine memory B cells from Bone Marrow and Spleen. |
| GSE140133 |
Single-Cell transcriptomes and BCR sequences of IgG+ murine memory B cells from Bone Marrow and Spleen |
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| Relations |
| BioSample |
SAMN13181003 |
| SRA |
SRX7087239 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4147195_spleen2_filtered_gene_bc_matrices_h5.h5 |
8.0 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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