TotalSeq™ Example Datasets

Cell Ranger 6.1.0 (10x Genomics)

Peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were stained separately with TotalSeq™ hashtags and the TotalSeq™-A Human Universal Cocktail, V1.0 and then pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation, and sequencing were carried out according to our protocol: TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol.

• 10,213 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode
• run with --expect-cells= 10,000

Cell Ranger 6.1.0 (10x Genomics)

Peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were stained separately with TotalSeq™ hashtags and the TotalSeq™-B Human Universal Cocktail, V1.0 and pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation and sequencing were carried out according to our protocol: TotalSeq™-B or -C with 10x Feature Barcoding Technology

• 10,132 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode
• run with --expect-cells= 10,000

Cell Ranger 6.0.2 (10x Genomics)

Peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were stained separately with TotalSeq hashtags and the TotalSeq™-C Human Universal Cocktail, V1.0 and pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation and sequencing were carried out according to our protocol: TotalSeq™-B or -C with 10x Feature Barcoding Technology.

• 12,062 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode
• run with --expect-cells= 10,000

Peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were activated with PMA and Ionomycin for 6 hours. For each donor, a total of two samples: the original resting cells, and the activated cells were collected. All four samples were stained with a 10% dilution of TotalSeq™-A Human Universal Cocktail, V1.0.

Staining, library preparation, and sequencing were carried out according to our protocol: Bulk Epitope and Nucleic Acid Sequencing (BEN-seq) Protocol

30 µL of the diluted libraries were sequenced to obtain paired end 30 bp (30x2) data on two MiniSeq High output 75 cycle kits (Illumina).

File Naming Scheme

Each file is named using the following template: d10x_pbmc_all_act_d1r1_ADT_S11_R1_001.fastq.gz, where:

• d10x refers to the samples being stained by 10x diluted TotalSeq™-A Human Universal Cocktail.
• PBMC refers to the cell type (peripheral blood mononuclear cells, or PBMC).
• all act refers to the sequenced sample contains 100% activated cells. This field is omitted for resting cells.
• d1 refers to the cells come from Donor 1.
• r1 refers to the sample being first of two replicates.
• ADT refers to the library sequenced is from antibody-derived tags (ADT).

The remaining fields are identical to the fields used given by bcl2fastq to demultiplexed FASTQ data files.

Cell Ranger 6.1.0 (10x Genomics)

Splenocytes harvested by mechanical dissociation from three C57BL/6 mice were stained separately with TotalSeq™ Hashtags and the TotalSeq™-A Mouse Universal Cocktail, V1.0 and pooled prior to GEM generation. Only resting cells in this analysis.

Staining, library preparation, and sequencing was carried out according to our protocol: TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol

• 16,689 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode
• run with --expect-cells= 10,000

Cell Ranger 6.1.0 (10x Genomics)

Splenocytes harvested by mechanical dissociation from three C57BL/6 mice were stained separately with TotalSeq™ hashtags and the TotalSeq™-B Mouse Universal Cocktail, V1.0 then pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation, and sequencing were carried out according to our protocol: TotalSeq™-B or -C with 10x Feature Barcoding Technology.

• 18,290 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp i7 sample barcode
• run with --expect-cells= 10,000

Cell Ranger 6.1.0 (10x Genomics)

Splenocytes harvested by mechanical dissociation from three C57BL/6 mice were stained separately with TotalSeq™ Hashtags and the TotalSeq™-C Mouse Universal Cocktail, V1.0 and then pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation, and sequencing were carried out according to our protocol: TotalSeq™-B or -C with 10x Feature Barcoding Technology.

• 13,566 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO). 
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp i7 sample barcode
• run with --expect-cells= 10,000

Cell Ranger 7.1.0 (10x Genomics)

Splenocytes were harvested by enzymatic dissociation of spleens from two C57BL/6 mice. T and B cell populations were depleted using MojoSort™ anti-PE Nanobeads, PE anti-mouse CD90.2 (Thy-1.2), and PE anti-mouse CD19. Cells from each mouse were stained separately with TotalSeq™ Hashtags, the TotalSeq™-B Mouse Myeloid Cocktail, V1.0, and then pooled prior to GEM generation. Only resting cells were included in this analysis.

Staining, library preparation, and sequencing was carried out according to our protocol: TotalSeq™-B or -C with 10x Feature Barcoding Technology.

• 10,022 cells detected
• Sequenced on Illumina NovaSeq 6000 with approximately 36,000 reads per cell (25,000 RNA, 10,000 ADT, 1,000 HTO).
• 28bp read 1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode
• run with --expect-cells= 10,000