Peripheral blood mononuclear cells (PBMCs) from a healthy donor were obtained by 10x Genomics from AllCells. 500,000 cells were stained with TotalSeq™-B Human Universal Cocktail, V1.0 (BioLegend, Cat# 399904) following the demonstrated protocol Cell Surface Protein Labeling for Chromium Fixed RNA Profiling for Singleplex Samples with Feature Barcode technology (CG000529, Rev A), with the minor modification that the staining was done in a total of 50μL (BioLegend recommendation for TotalSeq™ Universal Cocktails). After staining, cells were washed using the 2-Wash option and then fixed for 1 hour at room temperature following the demonstrated protocol Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling (CG000478).

Fixed RNA Gene Expression and Cell Surface Protein libraries were generated as described in the Chromium Fixed RNA Profiling for Singleplexed Samples with Feature Barcode technology for Cell Surface Protein User Guide (CG000477) using the Chromium X and sequenced on an Illumina NovaSeq 6000 with approximately 37,000 read pairs per cell.

Paired-end, dual indexing:

- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2


FASTQs

Multi Config CSV

Feature Reference CSV

Target Panel CSV

Sample Images

Loupe Browser visualization file (CLOUPE, raw)

Gene Expression - Feature / cell matrix (raw)

Gene Expression - Feature / cell matrix HDF5 (raw)

Gene Expression - Raw per-molecule read information

Gene Expression - Genome-aligned unassigned BAM

Gene Expression - Genome-aligned unassigned BAM index

Multiplexing Analysis - Tag assignment confidence (CSV)

Multiplexing Analysis - Cells per Tag (JSON)

Multiplexing Analysis - Number of Calls per Cell (CSV)

Multiplexing Analysis - Tag Call Summary (CSV)

VDJ Ig - All contig annotations (BED)

VDJ Ig - All contig annotations (CSV)

VDJ Ig - All contig annotations (JSON)

VDJ Ig - Read-contig alignments (BAM)

VDJ Ig - Read-contig alignment index (BAI)

VDJ Ig - All-contig FASTA

VDJ Ig - All-contig FASTA index (FAI)

VDJ Ig - All-contig FASTQ

VDJ TCR - All contig annotations (BED)

VDJ TCR - All contig annotations (CSV)

VDJ TCR - All contig annotations (JSON)

VDJ TCR - Read-contig alignments (BAM)

VDJ TCR - Read-contig alignment index (BAI)

VDJ TCR - All-contig FASTA

VDJ TCR - All-contig FASTA index (FAI)

VDJ TCR - All-contig FASTQ

VDJ reference

Multiplexing Analysis - FRP GEM Barcode Overlap (CSV)

Gene Expression - Probe barcode matrix HDF5 (raw)

Inputs/Library

Summary HTML

Summary CSV

Gene Expression - Loupe Browser file

Gene Expression - Genome-aligned BAM

Gene Expression - Genome-aligned BAM index

Gene Expression - Sample barcodes

Gene Expression - Feature / cell matrix HDF5 (per-sample)

Gene Expression - Feature / cell matrix (per-sample)

Gene Expression - Per-molecule read information

Gene Expression - Feature Reference

Gene Expression - Clustering analysis

Gene Expression - CRISPR analysis

Gene Expression - Target Panel

VDJ Ig - AIRR rearrangement (TSV)

VDJ Ig - Cell Barcodes (JSON)

VDJ Ig - Clonotype info (CSV)

VDJ Ig - Concatenated Segments (BAM)

VDJ Ig - Concatenated Segments index (BAI)

VDJ Ig - Concatenated Segments (FASTA)

VDJ Ig - Concatenated Segments index (FAI)

VDJ Ig - Clonotype consensus annotations (CSV)

VDJ Ig - Clonotype consensus BAM

VDJ Ig - Clonotype consensus index BAI

VDJ Ig - Clonotype consensus FASTA

VDJ Ig - Clonotype consensus index FAI

VDJ Ig - Filtered contig annotations (CSV)

VDJ Ig - Filtered contig sequences FASTA

VDJ Ig - Filtered contig sequences FASTQ

VDJ Ig - All contig info (ProtoBuf)

VDJ Ig - Loupe V(D)J Browser file

VDJ TCR - AIRR rearrangement (TSV)

VDJ TCR - Cell Barcodes (JSON)

VDJ TCR - Clonotype info (CSV)

VDJ TCR - Concatenated Segments (BAM)

VDJ TCR - Concatenated Segments index (BAI)

VDJ TCR - Concatenated Segments (FASTA)

VDJ TCR - Concatenated Segments index (FAI)

VDJ TCR - Clonotype consensus annotations (CSV)

VDJ TCR - Clonotype consensus BAM

VDJ TCR - Clonotype consensus index BAI

VDJ TCR - Clonotype consensus FASTA

VDJ TCR - Clonotype consensus index FAI

VDJ TCR - Filtered contig annotations (CSV)

VDJ TCR - Filtered contig sequences FASTA

VDJ TCR - Filtered contig sequences FASTQ

VDJ TCR - All contig info (ProtoBuf)

VDJ TCR - Loupe V(D)J Browser file

Gene Expression - Antibody analysis

Gene Expression - Probe Set (CSV)

Gene Expression - Raw feature / cell matrix HDF5 (per-sample)

Gene Expression - Raw feature / cell matrix (per-sample)

Gene Expression - Raw probe barcode matrix (per-sample)

Summary

PBMCs

Downsampled Library (GEX only)

Downsampled Sample Outputs (GEX only)

Chromium X Series

Chromium

Gene Expression

Epigenomic Profiling

Immune Profiling

Protein co-detection

Advanced system for multiomic analysis of hundreds to millions of cells.

Plate-based sample handling brings unprecedented scale to highly sensitive single cell profiling

Flex

Fixed RNA Profiling assay for comprehensive probe-based gene expression profiling with single cell resolution.

Cell Ranger

Chromium Single Cell Gene Expression provides single cell transcriptome 3' gene expression and multiomic capabilities to profile tens of thousands of cells. Explore cellular heterogeneity, novel targets, and biomarkers with combined gene expression, cell surface protein expression, or CRISPR edits in each cell.

Cell Surface Protein

CRISPR Screening

Sample multiplexing

Reveal the full complexity of cellular diversity, <em>cell by cell</em>

Single Cell Gene Expression

3' gene expression profiling at scale with single cell resolution.

Chromium Single Cell Immune Profiling provides a multiomic solution to your immunology questions. Analyze full-length V(D)J sequences for paired B-cell or T-cell receptors, cell surface protein expression, antigen specificity, and/or CRISPR edits, along with gene expression, all from a single cell.

Antigen Specificity

Multiomic profiling of immune diversity, <em>cell by cell</em>

Single Cell Immune Profiling

5' gene expression alongside V(D)J repertoire profiling and antigen specificity of T and B cells.

Profile a defined set of transcripts from single cells or tissue sections with Targeted Gene Expression. Reduce costs while increasing the number of samples, or increasing sequencing depth with customizable, comprehensive gene panels.

Discontinuation of Targeted Gene Expression

Chromium Connect

Automated system for single cell partitioning, barcoding, and library preparation.

Chromium Controller

Compact system for multiomic analysis of hundreds to tens of thousands of single cells.

Targeted Gene Expression

Visium

Profiling of defined transcript sets from single cells or fresh frozen tissue sections.

Analyze single cell gene expression data with a set of free, easy-to-use analysis pipelines.

10k Human PBMCs Stained with TotalSeq™-B Human Universal Cocktail, Singleplex Sample (Next GEM)

Chromium Fixed RNA Profiling offers comprehensive, scalable solutions to measure gene expression in formaldehyde fixed samples. This workflow allows for fixed samples to be processed and analyzed individually. This protocol also optionally enables the detection of cell surface proteins in fixed single cell suspensions that are labeled with antibody-oligonucleotide conjugates prior to fixation.\
\
For use with:

- Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns PN-1000414
- Chromium Next GEM Chip Q Single Cell Kit, 48 rxns PN-1000418 / 16 rxns PN-1000422
- Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 1 BC PN-1000474
- Chromium Fixed RNA Kit, Mouse Transcriptome, 4 rxns x 1 BC PN-1000495
- Fixed RNA Feature Barcode Kit 16 rxns PN-1000419
- Dual Index Kit TT Set A 96 rxns PN-1000215
- Dual Index Kit TS Set A 96 rxns PN-1000251

<SupportCallout level={"warning"}>{"Optional cell surface protein labeling must be completedprior to sample fixation."}</SupportCallout>

<SupportCallout level={"warning"}>{"Firmware FW 1.1.0 or higher on Chromium X/iX is required for running this assay. Chip Q is only compatible with Chromium X/iX."}</SupportCallout>


Provides instructions for constructing Singleplexed Gene and (TotalSeq™️-B) Protein Expression libraries.

Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples With Feature Barcode technology for Protein

Chromium Fixed RNA Profiling (Gene Expression Flex) with Feature Barcode technology offers comprehensive, scalable solutions to measure gene and protein expression in formaldehyde fixed samples. To measure protein expression, cell surface and intracellular proteins are labeled using an antibody conjugated to a Feature Barcode oligonucleotide, followed by a fixation with formaldehyde to ensure 1) the cells are properly fixed and permeabilized for probe hybridization and 2) the antibodies firmly bind to the cells, preventing their loss during probe hybridization.

This document outlines cell surface and intracellular protein labeling protocols for use with Chromium Fixed RNA Profiling. General guidance on antibody-oligonucleotide conjugation, evaluating antibodies with flow cytometry, and best practices when staining cells is also provided here.

<SupportCallout level={"warning"}>{"Optional cell surface protein labeling must be completed prior to sample fixation."}</SupportCallout>

<SupportCallout level={"warning"}>{"TotalSeq™️-C antibodies are compatible with Singleplex and Multiplex workflows (via Barcode Oligo Capture); TotalSeq™️-B antibodies are only compatible with Singleplex workflow."}</SupportCallout>


Outlines cell surface protein and intracellular labeling use and guidance for enriching labeled cells using Fluorescence Activated Cell Sorting (FACS).

Cell Surface & Intracellular Protein Labeling for Chromium Fixed RNA Profiling

10k Human PBMCs Stained with TotalSeq™-B Human Universal Cocktail, Singleplex Sample (Next GEM)

Assess data quality

Visualize and explore data