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| Status |
Public on Mar 01, 2024 |
| Title |
P40.L109.day23 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
patient: P40
Sex: F
condition: SLE
responder: NR
time point: day23
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| Extracted molecule |
total RNA |
| Extraction protocol |
Live frozen PBMCs were thawed and washed twice with RPMI supplemented with 10% FBS and 20 μg ml−1 DNase I (Sigma Aldrich). Total PBMCs (1 million cells) were stained with a cocktail of TotalSeq-A antibodies (BioLegend) in PBS supplemented with 5% FBS, 2 mM EDTA and 5 mg ml−1 human IgG, washed twice with PBS supplemented with 5% FBS, and 2 mM EDTA, and resuspended in PBS supplemented with 1% BSA (Miltenyi Biotec), and 0.5 U μl−1 RNase Inhibitor (Sigma Aldrich). Approximately 9,000 cells were targeted for each experiment. Cells were mixed with the reverse transcription mix and subjected to partitioning along with the Chromium gel-beads using the 10X Chromium system to generate the gel-bead in emulsions (GEMs) using the 3′V3 chemistry (10X Genomics). The reverse transcription reaction was conducted in the C1000 touch PCR instrument (BioRad).
Barcoded cDNA was extracted from the GEMs by post-GEM reverse transcription cleanup and amplified for 12 cycles. Before amplification, the cDNA amplification mix was spiked in with ADT additive primer (0.2 μM stock) to amplify the antibody barcodes. Amplified cDNA was subjected to 0.6× SPRI beads cleanup (Beckman, B23318). Amplified antibody barcodes were recovered from the supernatant and were processed to generate TotalSeq-A libraries as instructed by the manufacturer (BioLegend, TotalSeq-A antibodies with 10x Single Cell 3′ Reagent Kit v.3 3.1 protocol). The rest of the amplified cDNA was subjected to enzymatic fragmentation, end repair, A tailing, adaptor ligation and 10X-specific sample indexing as per manufacturer’s protocol. Libraries were quantified using Bioanalyzer (Agilent) analysis. 10x Genomics scRNA-seq and TotalSeq-A libraries were pooled and sequenced on an Illumina HiSeq 4000 using the recommended sequencing read lengths of 28 bp (read 1), 8 bp (i7IndexRead) and 91 bp (read 2).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
patient40, day23
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| Data processing |
CellRanger v.3.1.0 (10xGenomics) was used to demultiplex raw sequencing data and quantify transcript levels against the 10x Genomics GRCh38 reference v.3.0.0.
Assembly: GRCh38
Supplementary files format and content: 1) matrix.mtx.gz: matrix containg read count; 2) barcodes.tsv.gz: cell names; 3) features.tsv.gz: gene/features names
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| Submission date |
Dec 12, 2023 |
| Last update date |
Mar 01, 2024 |
| Contact name |
Hong Zheng |
| Organization name |
Stanford university
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| Street address |
240 Pasteur Dr
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94304 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE250024 |
Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus |
| GSE250025 |
Impaired innate and adaptive immune responses to BNT162b2 SARS-CoV-2 vaccination in systemic lupus erythematosus |
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| Relations |
| BioSample |
SAMN38796428 |
| SRA |
SRX22873400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7971532_L109_7Oct.barcodes.tsv.gz |
4.9 Kb |
(ftp)(http) |
TSV |
| GSM7971532_L109_7Oct.features.tsv.gz |
326.4 Kb |
(ftp)(http) |
TSV |
| GSM7971532_L109_7Oct.matrix.mtx.gz |
9.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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