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Sample GSM5449998

Query DataSets for GSM5449998

Status

Public on Jun 29, 2022

Title

Group A - Patient 1-Feature

Sample type

SRA

Source name

Human PBMCs

Organism

Homo sapiens

Characteristics

tissue: Blood
cell type: PBMC
treatment: Immune Checkpoint Inhibitor
disease state: No irAE
molecule subtype: Surface Protein Feature Barcode Sequence

Extracted molecule

total RNA

Extraction protocol

Frozen PBMCs were removed from liquid nitrogen and resuspended in warm RPMI media + 10% FBS (@ 37ºC) by adding 1 mL, 3 mL, 4 mL, 8 mL 16 mL media at 30-60 second intervals. Cells were washed and passed through a 40 um filter, centrifuged @ 300 rcf x 5 min and resuspended in PBS with 50 ul 1% BSA and Biolegend Fc Receptor Blocking Solution (Human TrueStain FcX (Cat. No. 422301) for 20 minutes. Afterwards, another 50 uL of antibody mastermix containing Biolegend TotalSeq-C antibodies for staining (Biolegend TotalSeq™-C0063 anti-human CD45RA Antibody Cat. No. 304163; Biolegend TotalSeq™-C0072 anti-human CD4 Antibody Cat. No. 300567; Biolegend TotalSeq™-C0046 anti-human CD8 Antibody Cat. No. 344753) for final manufacture-recommended concentration of 1.0 ug of antibody in 100 ul of staining buffer for every 1 million cells for a total of 10-15 minutes incubation. After incubation, cells were washed 2x by resuspending in 1.5 mL PBS + 1% RNase-free BSA (MACS BSA Stock Solution No. 130-091-376) and then 1 mL PBS + 0.04% BSA. 10 uL of cell suspension was then taken for counting and appropriate volume of PSA + 0.04% BSA was added (200-500 uL) to aim for final target concentration of 1 million cells/mL). Cells were then loaded into Chromium Next GEM Chip G (10x PN 2000177) to achieve 6000-8000 target cell recovery per sample according to the manufacture’s protocol.
All steps from cDNA isolation to library preparation were completed according to 10x’s manufacturing protocols for the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1. Isolated cDNA was amplified (14 cycles), and then cDNA was allocated for TCR enrichment, feature barcode library generation, and 5’ cDNA library generation. cDNA and library quality was evaluated using the high sensitivity DNA kit on the Agilent 2100 Bioanalyzer.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

CITE-seq data
Processed data included on GSM5449974
A1-filtered_feature_bc_matrix.tar
A1-Feature

Data processing

Library strategy: CITE-seq
Raw single-cell RNA-seq data were processed using 10x Genomics Cell Ranger (4.0.3) to demultiplex the FASTQ reads in order to align them to the human reference genome (GRCh38, v4.0.0, from 10X Genomics) and count the unique molecular identifier (UMI) (through the “cellranger count” function).
Raw T cell receptor (TCR) sequencing data were processed using 10x Genomics Cell Ranger (v4.0.3). In summary, the algorithm aligned FASTQ reads to human GRCh38 V(D)J reference genome (v4.0.0, from 10X Genomics) by using the “cellranger vdj” function, resulting in the assembly of V(D)J sequences and clonotypes (38).
Individual sample gene expression matrices were loaded into Seurat v4.0.0 R package (https://satijalab.org/seurat/) for further analysis.
To filter out poor quality data from cells with high mitochondrial gene expression likely to be dead/damaged cells (59), we removed cells with greater than >15% mitochondrial genes present (60). Additionally, we removed cells for which less than 500 genes were detected.
Genome_build: h38
Supplementary_files_format_and_content: Cell Ranger output

Submission date

Jul 13, 2021

Last update date

Jul 18, 2022

Contact name

Han Zhu

E-mail(s)

hanzhu@stanford.edu

Organization name

Stanford University

Department

Cardiovascular Medicine