10x Genomics Single-Cell Processing

 

The 10x Genomics Chromium Controller is a single-cell profiling technology that enables the analysis of large cell numbers at a high capture efficiency (of up to 65%). The platform allows for high-throughput analysis in a variety of cell types as well as single-cell nuclei. The workflow encapsulates cells or nuclei together with gel beads into nanodroplets (single-Poisson distribution loading). For most assays, up to eight samples can be processed per run allowing for the capture of up to 80,000 cells per run. Trained core staff will prepare the libraries from single-cell suspensions submitted by the research investigator.

Image provided by 10x Genomics

Sample Requirements:

  • Cell Viability: >80%
  • Cell concentration: 750-1200 cells/μl
  • Total volume: 50 μl 

Book an appointment! Detailed sample submission instructions can be downloaded here. Single-cell data are analyzed with the Cell Ranger and Loupe Cell Browser software.

Helpful documents:

Cell Preparation Guide

Dead Cell Removal Protocol

Tumor Dissociation Protocol

 

 

The full list of 10X solutions are listed below:

Single Cell Gene Expression (3′ GEX)

Single cell expression measurements that enable discovery of gene expression dynamics and molecular profiling of individual cell types.  

AGC scRNA V3.1 (3′ GEX) Reagents Aug 2020 – present

Vendor Part Number Description
10x Genomics 1000268 Chromium Next GEM Single Cell 3′ Kit v3.1, 16 rxns
10x Genomics 1000120 Chromium Next GEM Chip G Single Cell Kit, 48 rxns
10x Genomics 1000215 Dual Index Kit TT Set A 96 rxns

Note – The Single Index Kit T Set A, 96 rxn (1000213) was used with the V3.1 reagents July 2019 – August 2020.

Demonstrated sample processing protocols can be found on the 10X Single Cell Gene Expression Sample Prep page.

Allowing up to 1000 cells/nulcei per capture, the 3′ Gene Expression – Low Throughput (3′ GEX LT) solution is an excellent option to collect pilot data or test you cell and/or nuclei isolation protocols.

Vendor Part Number Description
10x Genomics 1000325 Chromium Next GEM Single Cell 3′ LT Kit v3.1, 4 rxns

 

Single Cell Immune Profiling (VDJ and 5′ GEX)

Profiling full-length paired V(D)J transcripts from lymphocytes on a cell-by-cell basis. Can be coupled with 5’ gene expression measurements to provide high resolution insights into the adaptive immune system. 

AGC Immune Profiling  V2 Reagents September 2020 – present

Vendor Part Number Description
10x Genomics 1000263 Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit V2, 16 rxns
10x Genomics 1000286 Chromium Next GEM Chip K Single Cell Kit, 48 rxns
10x Genomics 1000253 Single Cell Human BCR, 16 rxns
10x Genomics 1000252 Single Cell Human TCR, 16 rxns
10x Genomics 1000255 Single Cell Mouse BCR, 16 rxns
10x Genomics 1000254 Single Cell Mouse TCR, 16 rxns
10x Genomics 1000215 Dual Index Kit TT Set A, 96 rxns
10x Genomics 1000256 5′ Feature Barcode Kit, 16 rxns
10x Genomics 1000250 Dual Index Kit TN Set A, 96 rxns

Demonstrated sample processing protocols can be found on the 10X Single Cell Immune Profiling Sample Prep page.

Single Cell Assay for Transposase Accessible Chromatin (ATAC)

Genome-wide chromatin accessibility information for elucidating gene regulatory mechanisms to understand epigenetic heterogeneity.

AGC ATAC V1.1 Reagents July 2019 – present

Vendor Part Number Description
10x Genomics 1000175 Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1, 16 rxns
10x Genomics 1000161 Chromium Next GEM Chip H Single Cell Kit, 48 rxns
10x Genomics 1000212 Single Index Kit N Set A, 96 rxn

Demonstrated sample processing protocols can be found on the 10X Single Cell ATAC Sample Prep page.

Single Cell Multiome (3’GEX + ATAC)

Simultaneously profile gene expression and open chromatin from the same cell to characterize cell types, identify states, and uncover gene regulatory programs.

Vendor Part Number Description
10x Genomics 1000283 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit, 16 rxns
10x Genomics 1000234 Chromium Next GEM Chip J Single Cell kit, 48 rxns
10x Genomics 1000215 Dual Index Kit TT Set A 96 rxns
10x Genomics 1000212 Single Index Kit N Set A, 96 rxns

Demonstrated nuclei prep protocols can be found on the 10X Single Cell Multiome Sample Prep page.

Feature Barcoding (CITE-seq and Cell Multiplexing)

Feature Barcoding is the broad term used by 10X Genomics in order to describe any method by which cell surface features can be used to gain useful biological information. Mostly this comes in two forms – looking for cells that are expressing certain proteins on the cell surface (CITE-seq) or using ubiquitous markers in order to pool different samples together into one channel of the chip (Cell Multiplexing/Cell Hashing).

As part of their TotalSeq antibody catalog, BioLegend offers this technology through antibody-oligonucleotide conjugates that can be used for both CITE-seq and Cell Hashing. Antibodies come in three formats that differ in their capture sequence for library preparation so pay close attention to which antibody catalog you use. These are limited to Human and Mouse samples.

  • TotalSeq A is compatible with any Single Cell RNA-seq product that uses poly(dT) for mRNA capture. This is the format originally used in the CITE-seq protocol. At this time, the 10X Genomics analysis pipeline (CellRanger) does not support the processing of the protein derived libraries. Data must be analyzed using the R based Searat package.
  • TotalSeq B is specifically designed to be compatible with the newer 10X Genomics Single Cell 3’ v3  reagents used in the core and is fully supported by the 10x Genomics CellRanger software. These antibody oligos are complementary to a unique capture sequence on the Single Cell 3′ v3 Gel Beads so antibody barcodes do not compete for the oligo(dT)s capturing the mRNA.
  • TotalSeq C is specifically designed to be compatible with the 10X Single Cell 5′ workflow used in the Immune Profiling kit. It is also fully supported by 10x Genomics CellRanger software run.

If using 10X Genomics single cell 3′ GEX, TotalSeq B antibodies and/or hashtags are the best way to go! 

10x Genomics has a method by which cells can be multiplexed together is a single channel of the chip that they refer to as Cell Multiplexing Oligos. This method uses lipid-oligonucleotide complexes in order to label cells with a distinct oligo. This method is species agnostic and allows the user to capture a maximum of 30,000 cells in a single channel. It is also fully supported by the 10x pipeline and tech support. Cellplex reagents can be purchased from the core.

Image provided by 10x Genomics

Vendor Part Number Description
10x Genomics 1000261 3′ CellPlex Kit Set A, 48 rxns
10x Genomics 1000262 3′ Feature Barcode for CellPlex
10x Genomics 1000243 Dual Index Kit NN Set A, 96 rxns

Frequently Asked Questions

 

How many cells should I target for capture and how many reads/cells do I need?

This 10X Genomics Technical Note discusses how sequencing depth and cell number influence the detection of major cell types in peripheral blood mononuclear cells (PBMCs).

Can I have my cells checked without running my full experiment?

Yes, we offer viability only appointment slots for you to check your cell dissociation method before using experimental cells. Select “viability/count only” as your assay type when filling out the MiCores 10X appointment request form. We ask that you bring a maximum of four samples for the appointment.

How much sequencing do my samples need?

The 10X minimum recommendations are as follows:

  • ATAC – 25,000 reads/cell
  • Gene Expression (3’ and 5’) – 20,000 reads/cell
  • Immune Profiles (B and T cells) – 5,000 reads/cell
  • ADTs and HTOs – 5,000 reads/cell
What sort of buffers can my samples be in?

This is dependent on which analysis you would like to do.

  • ATAC – Your nuclei must be in 1X Nuclei Buffer from 10X Genomics. We have this buffer available for you to pick up from us prior to your appointment time.
  • CNV (Single Cell DNA/Copy Number Variation) – Cells must be in a buffer that contains no more than 0.04% BSA or 10% FBS. Supported buffers and media include: PBS+0.04%BSA, HBSS+0.04%BSA, EMEM+10%FBS, DMEM+10%FBS, RPMI+10%FBS, Hams F12+10%FBS, IMDM+10%FBS, DMEM:F12+10%FBS
  • Gene Expression and Immune Profiling – Both of these assays are compatible with a wide variety of buffers and media. Some examples are: EMEM+10%FBS, DMEM+10%FBS, RPMI+10%FBS, Hams F12+10%FBS, DMEM:F12+10%FBS, M199.
Can I FACS sort my cells before I bring them?

Absolutely. Some things to keep in mind when sorting your cells prior to bringing them:

  • FACS will overestimate the number of cells on average by 50%. One way to mitigate this problem is to stain your cells with Ruby. This will separate the cells from debris and make the count much more accurate.
  • Your cells may not like to stay in FACS buffer. You can sort into a full media that is compatible with your cells, and resuspend them in a compatible media afterward.
  • Your cells will likely be very dilute when they come out of the sorter. You will likely want to concentrate them before coming to the Core.
Can we do CITE-seq or cell hashing?

Yes, we support CITE-seq (ADT) and cell hashing (HTO) libraries. 10X Genomics refers to these as Feature Barcoded libraries. We primarily work with the BioLegend TotalSeq suite of antibodies, but are willing to work with your project. One recommendation is that if you are FACS sorting, simultaneously stain your cells with the FACS antibodies as well as the single cell antibodies.

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