DOI: 10.5281/zenodo.13971562
title: Single-cell RNA-Seq and TCR-Seq analysis of PD-1+ CD8+ T-cells responding to anti-PD-1 and anti-PD-1/CTLA-4 immunotherapy in melanoma

description: This dataset details the scRNASeq and TCR-Seq analysis of sorted PD-1+ CD8+ T cells from patients with melanoma treated with checkpoint therapy (anti-PD-1 monotherapy and anti-PD-1 & anti-CTLA-4 combination therapy) at baseline and after the first cycle of therapy. A major publication using this dataset is accessible here: (reference)   

 

*experimental design

 Single-cell RNA sequencing was performed using 10x Genomics with feature barcoding technology to multiplex cell samples from different patients undergoing mono or dual therapy so that they can be loaded on one well to reduce costs and minimize technical variability. Hashtag oligomers (oligos) were obtained as purified and already oligo-conjugated in TotalSeq-C format from BioLegend. Cells were thawed, counted and 20 million cells per patient and time point were used for staining. Cells were stained with barcoded antibodies together with a staining solution containing antibodies against CD3, CD4, CD8, PD-1/IgG4 and fixable viability dye (eBioscience) prior to FACS sorting. Barcoded antibody concentrations used were 0.5 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. After staining, cells were washed twice in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 xg 5 min at 4 °C) and supernatant exchange. After the final wash, cells were resuspended in PBS and filtered through 40 µm cell strainers and proceeded for sorting. Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions. Gene expression, hashing and TCR libraries were pooled to desired quantities to obtain the sequencing depths of 15,000 reads per cell for gene expression libraries and 5,000 reads per cell for hashing and TCR libraries. Libraries were sequenced on a NovaSeq 6000 flow cell in a 2X100 paired-end format.

 

*extract protocol

 PBMCs were thawed, counted and 20 million cells per patient and time point were used for staining. Cells were stained with barcoded antibodies together with a staining solution containing antibodies against CD3, CD4, CD8, PD-1/IgG4 and fixable viability dye (eBioscience) prior to FACS sorting. Barcoded antibody concentrations used were 0.5 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. After staining, cells were washed twice in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 xg 5 min at 4 °C) and supernatant exchange. After the final wash, cells were resuspended in PBS and filtered through 40 µm cell strainers and proceeded for sorting. Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions.

 

*library construction protocol

 Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions. Gene expression, hashing and TCR libraries were pooled to desired quantities to obtain the sequencing depths of 15,000 reads per cell for gene expression libraries and 5,000 reads per cell for hashing and TCR libraries. Libraries were sequenced on a NovaSeq 6000 flow cell in a 2X100 paired-end format.

 

*library strategy

 scRNA-seq and scTCR-seq

 

*data processing step

 Pre-processing of sequencing results to generate count matrices (gene expression and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline.

 Further processing was done with Seurat (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis based on gene expression).

 

 *genome build/assembly

 Alignment was performed using prebuilt Cell Ranger human reference GRCh38.

 

*processed data files format and content

 RNA counts and HTO counts are in sparse matrix format and TCR clonotypes are in csv format.

Datasets were merged and analyzed by Seurat and the analyzed objects are in rds format.

 






file name



file checksum





PD1CD8_160421_filtered_feature_bc_matrix.zip



da2e006d2b39485fd8cf8701742c6d77





PD1CD8_190421_filtered_feature_bc_matrix.zip



e125fc5031899bba71e1171888d78205





PD1CD8_160421_filtered_contig_annotations.csv



927241805d507204fbe9ef7045d0ccf4





PD1CD8_190421_filtered_contig_annotations.csv



8ca544d27f06e66592b567d3ab86551e





  






*processed data file 



antibodies/tags





PD1CD8_160421_filtered_feature_bc_matrix.zip



none





PD1CD8_160421_filtered_feature_bc_matrix.zip



TotalSeq™-C0251 anti-human Hashtag 1 Antibody - (HASH_1) - M1_base_monotherapyTotalSeq™-C0252 anti-human Hashtag 2 Antibody - (HASH_2) - M1_post_monotherapyTotalSeq™-C0253 anti-human Hashtag 3 Antibody - (HASH_3) - C1_base_combined_therapyTotalSeq™-C0254 anti-human Hashtag 4 Antibody - (HASH_4) - C1_post_combined_therapyTotalSeq™-C0255 anti-human Hashtag 5 Antibody - (HASH_5) - C2_base_combined_therapyTotalSeq™-C0256 anti-human Hashtag 6 Antibody - (HASH_6) - C2_post_combined_therapy





PD1CD8_160421_filtered_contig_annotations.csv



none





PD1CD8_190421_filtered_feature_bc_matrix.zip



none





PD1CD8_190421_filtered_feature_bc_matrix.zip



TotalSeq™-C0251 anti-human Hashtag 1 Antibody - (HASH_1) - M2_base_monotherapyTotalSeq™-C0252 anti-human Hashtag 2 Antibody - (HASH_2) - M2_post_monotherapyTotalSeq™-C0253 anti-human Hashtag 3 Antibody - (HASH_3) - M3_base_monotherapyTotalSeq™-C0254 anti-human Hashtag 4 Antibody - (HASH_4) - M3_post_monotherapyTotalSeq™-C0255 anti-human Hashtag 5 Antibody - (HASH_5) - C3_base_combined_therapyTotalSeq™-C0256 anti-human Hashtag 6 Antibody - (HASH_6) - C3_post_combined_therapy





PD1CD8_190421_filtered_contig_annotations.csv



none