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| Status |
Public on Jun 11, 2021 |
| Title |
Group4_GEX |
| Sample type |
SRA |
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| Source name |
Activated T and B cells from four patients vaccinated against COVID19 after kidney transplantation with Biotech/Pfizer 7 days after boost
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| Organism |
Homo sapiens |
| Characteristics |
individual: mixed sample (non-demultiplexed) representing four patients vaccinated against COVID19 after kidney transplantation
responder status: mixed sample (non-demultiplexed) representing three non-responding donors and one responding donor
tissue: peripheral blood
cell type: mixed sample (non-demultiplexed) representing FACS-sorted activated T cells (CD38+ HLA-DR+), memory B cells (CD38low CD27+) and plasmabolasts (CD38high CD27high)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were enriched from the peripheral blood by MACS using StraightFrom Blood CD3, CD19 and CD138 Microbeads. Cells were subsequently incubated with barcoded and fluorochorme-conjugated antibodies and sorted by FACS as live CD3+CD14-CD16-CD38+HLA-DR+, live CD3-CD14-CD16-CD38lowCD27+ and live CD3-CD14-CD16-CD38highCD27high. The barcoded antibodies used for cell-surface protein analysis were anti-human CD138 (GTATAGACCAAAGCC), CD20 (TTCTGGGTCCCTAGA), CD4 (GAGGTTAGTGATGGA), CD8 (GCGCAACTTGATGAT), CD45RO (CTCCGAATCATGTTG), CD45RA (TCAATCCTTCCGCTT), CD25 (TTTGTCCTGTACGCC), CD127 (GTGTGTTGTCCTATG) and CD154 (GCTAGATAGATGCAA). Sorted populations from three non-reponding and one reponding donor were pooled in equal proportions and applied to the 10X Genomics platform using the Single Cell 5’ Library & Gel Bead Kit (10x Genomics) following the manufacturer’s instructions.
The purified cDNA was amplified using 12 cycles of PCR and was subsequently used for 5’ gene expression (GEX), cell-surface protein, TCR and BCR library preparation acording to 10x Genomics Chromium Next Gem Single Cell V(D)J Reagent Kits v1.1 with Feature Barcoding technology for Cell Surface Protein. The quality of the libraries was assessed by Qubit quantification and Bioanalyzer fragment analysis (Hight Sensitivity DNA Kit, Agilent).
The sequencing was performed on a NextSeq2000 device (Illumina) using P3 reagent kit (200 cycles) with the recommended sequencing conditions for 5’ GEX and cell-surface protein libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and P3 reagent kit (300 cycles) for TCR and BCR libraries (read1: 151nt, read2: 151nt, index1: 8nt, index2: n.a., 2% PhiX spike-in).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
Group4.filtered_feature_bc_matrix.h5
Group4.filtered_response_annotation.txt
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| Data processing |
Barcoded BAM, with Chromium cellular and molecular barcodes for each read, stored as TAG fields.
The Cell Ranger Single Cell Software Suite 3.1.0 was used to perform sample demultiplexing, barcode processing, and single cell 5’ gene counting for transcriptome wirh refdata-cellranger-hg19-1.2.0 as reference and expected-cells number of 3000 for each sample; polymorphisms removed.
For immune profiling Cell Ranger Single Cell Software Suite 3.1.0 was used for demultiplexing, barcode processing and assembly of the TCR as well as BCR sequences, using the vdj command and refdata-cellranger-vdj-GRCh38-alts-ensembl-2.0.0 as reference.
Genome_build: hg19
Supplementary_files_format_and_content: BAM comprising h5: aggregated gene count matrix in hdf5 format, including antibody UMI counts. fasta: high-confidence contig sequences of immune receptors with the corresponding cellular barcode and contig-id; csv: results from single cell immune profiling, comprising the annotation of the immune receptor, cellular barcode and contig-id; txt: tab delimited annotation of donors, comprising the cellular barcode and response after vaccination against COVID19.
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| Submission date |
Jun 09, 2021 |
| Last update date |
Jun 11, 2021 |
| 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 |
GPL30173 |
| Series (1) |
| GSE176442 |
Impaired antigen-specific memory B cell and plasma cell responses including lack of specific IgG upon SARS-CoV-2 BNT162b2 vaccination among Kidney Transplant and Dialysis patients |
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| Relations |
| BioSample |
SAMN19641103 |
| SRA |
SRX11099264 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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