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# Glossary of Terms

## Single Cell Gene Expression

GEM: Gel Beads-in-emulsion, an emulsion that contains a mixture of biochemistry reagents (uniquely barcoded gel beads) and zero, one, or more suspended cells/nuclei.

GEM well (formerly GEM group): A set of partitioned cells (Gel Beads-in-emulsion) from a single 10x Genomics Chromium™ Chip channel. One or more sequencing libraries can be derived from a GEM well.

Cell Barcode: The barcode associated with reads that is in cells.

Non-Cell Barcode: The barcode associated with reads that is outside cells (compared to "cell barcodes").

Library (or Sequencing Library): A 10x-barcoded sequencing library prepared from a single GEM well. With Feature Barcode or V(D)J assays, it is possible to create multiple libraries from the same GEM well. The library types may include Gene Expression, Antibody Capture, CRISPR Guide Capture, TCR-enrichment, etc.

LT (or Low Throughput): The Chromium Next GEM Single Cell 3’ LT v3.1 kit is a low throughput, cost-effective solution for smaller-scale and pilot studies for profiling whole transcriptome at the single cell level for 100 - 1,000 cells per sample. In combination with Feature Barcode technology (Antibody Capture), the assay also enables simultaneous cell surface protein detection in single cells.

HT (or High Throughput): The Chromium Next GEM Single Cell 3' HT v3.1 kit is a high throughput, cost-effective solution for profiling gene expression at the single cell level for 2,000-20,000 cells per channel or 2,000-60,000 cells per channel with 3' Cell Multiplexing. In combination with Feature Barcode technology, the assay also enables simultaneous cell surface protein detection or CRISPR profiling in single cells.

Sample: A cell suspension extracted from a single biological source (blood, tissue, etc).

Sequencing Run (or Flow cell): A flow cell containing data from one sequencing instrument run. The sequencing data can be further demultiplexed by lane or by sample indices.

## Feature Barcode Technology

Feature Barcode Antibody (or Antibody): Refers to a Feature Barcode reagent consisting of an antibody with high affinity to a known Cell Surface Protein coupled to a Feature Barcode oligonucleotide that identifies the antibody. These reagents are used to quantify the expression of cell surface proteins. For example, the TotalSeq™-B product line is a family of Feature Barcode antibodies that are compatible with the Single Cell 3' v3 solution.

Cell Surface Protein: A protein that is localized to the cell membrane, typically containing extracellular domains. These proteins can be quantified with Feature Barcodes such as TotalSeq antibody-oligonucleotide conjugates.

CRISPRa (or CRISPR activation): Similar to CRISPRi, but uses a Cas9 fused to an activating domain to promote expression of target gene instead of repressing it.

CRISPR Guide RNA: See sgRNA

CRISPRi (or CRISPR Interference): A method for measuring the impact of perturbations to gene expression levels. sgRNAs with protospacers targeting a gene of interest are used with a non-cutting Cas9 that is fused to a repressive domain. This represses the expression of the selected gene.

Count Matrix (or Feature-Barcode Matrix): Formerly known as the Gene-Barcode Matrix. A matrix of counts representing the number of unique observations of each feature within each cell barcode. Genes defined by the transcriptome reference and Feature Barcodes defined in the Feature Reference appear as rows in the matrix. Each barcode is a column of the matrix.

CROP-Seq: An assay scheme for pooled CRISPRi and CRISPRa experiments with single-cell Gene Expression readout. See Datlinger et al., Nature Methods 2017

Dextramer: Refers to a Feature Barcode reagent consisting of multiple copies of a peptide-MHC (p-MHC) complex conjugated to a Dextran backbone, coupled to a DNA oligonucleotide carrying a Feature Barcode that identifies the peptide-MHC complex. The p-MHC complex is the antigen of a T-Cell Receptor. Dextramers compatible with 10x Genomics Feature Barcode Technology are supplied by Immudex.

Feature: A unique type of countable molecule. Can refer to a gene, a barcoded antibody, a CRISPR Guide RNA or another barcoded reagent. Each feature is either a gene declared in the transcriptome reference or a feature barcode declared in the feature reference file. Corresponds to a row in the Count Matrix.

Feature Barcode: The subsequence of a Feature Barcode read that uniquely identifies the identity of the Feature Barcode reagent.

Feature Reference: A CSV file declaring the name, read layout, and barcode sequence of the all the Feature Barcode reagents in use in an experiment. A Feature Reference CSV must be provided to cellranger count when using Feature Barcode Technology. See the Feature Reference Documentation for details.

Guide RNA (or sgRNA, or Single Guide RNA): The Guide RNA, along with a Cas9 enzyme form the CRISPR system. The protospacer region of the Guide RNA recognizes a particular sequence in the genome.

p-MHC (or Peptide-MHC): An antigen-presenting MHC gene, bound to a displayed peptide. These complexes are recognized by T-Cell receptors in the adaptive immune system. Dextramers are Feature Barcode capable p-MHC reagent technology.

Perturb-Seq: The original demonstration of a pooled CRISPRi assay with a single-cell Gene Expression readout, using barcodes to identify which CRISPRi perturbations were present in each cell. See Dixit et al., Cell 2016.

## Targeted Gene Expression

Target Panel CSV file: A CSV file declaring the target gene panel used for a Targeted Gene Expression experiment, which specifies detailed information about the targeted genes and baits included in the panel. This file must be provided to cellranger count via the --target-panel option when analyzing Targeted Gene Expression data. Details and specifications of the Target Panel CSV file format are documented here.

Baits: A set of oligonucleotide sequences designed to specifically hybridize to and recover molecules from targeted genes during the hybridization-capture enrichment step of the Targeted Gene Expression assay.

WTA (or Whole Transcriptome Analysis): Whole Transcriptome Analysis, describes a (non-targeted) Chromium single-cell RNA-seq library or dataset which may be used as input for a Targeted Gene Expression experiment.

Parent: A WTA single-cell gene expression library or dataset that was used as the input material for a Targeted Gene Expression experiment. Because the Parent and its corresponding Targeted dataset are both derived from the same Library and/or GEM well, it is possible to directly compare results from these matched datasets in a pairwise manner, as enabled by cellranger targeted-compare.

## Cell Multiplexing

Cell Multiplexing: The labeling of a given cell (or nuclei) sample with a molecular tag and subsequently mixing this sample with other labeled samples. Introduced in Cell Ranger 6.0.

Cell Multiplexing Oligo (CMO): A specific type of feature barcode used to tag cells prior to pooling in a single GEM well.

Multiplet: A cell-associated barcode containing more than one cell. Multiplets that are assigned more than 1 CMO are detected and filtered out.

Physical Library: A sequencing library produced from a single GEM well.

Singlet: A cell-associated barcode assigned exactly one CMO. Only these are assigned to samples.

## Fixed RNA Profiling

10x GEM Barcode: The barcode associated with the 10x Genomics gel bead.

Probe Barcode: The unique barcode on the right hand side (RHS) probe.

Probe Set: A whole-transcriptome reference file declaring the gene panel used for a Fixed RNA Profiling experiment, which specifies detailed information about the genes which are targeted by each probe. This file must be provided to cellranger multi via the probe-set field in the multi config file.

Probe Filter: A column within the whole-transcriptome Probe Set reference file declaring the gene panel used for a Fixed RNA Profiling experiment. By default, probes predicted to have some off-target activity to homologous genes or sequences are excluded from analysis. Users can include UMI counts from all probes, including those with potential off-target activity, by setting the filter-probes field to false in the multi config file.

RNA-Templated Ligation: Fixed RNA Profiling is designed around a strict probe pairing framework. For each target locus, when two half-probe sequences bind to the proper locus and ligate together during the assay, a countable barcode-UMI-probe product is made.