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The Single Cell Core will be closed on Monday, September 5, according to the University approved Labor Day holiday.

Services

3’ Gene Expression
Measure gene activity on a cell-by-cell basis and characterize transcriptomes of complex cell populations.

 

What it Assays: The Chromium Single Cell Gene Expression Solution provides a comprehensive, scalable solution for cell characterization and gene expression profiling of hundreds to tens of thousands of cells. The latest improvements allow you to detect even more unique transcripts per cell, and with the addition of Feature Barcoding technology, you can get a more complete molecular readout cell by cell—identify cell-specific CRISPR-mediated perturbations or simultaneously measure gene and cell surface protein expression in the same cell, with virtually unlimited possibilities for the additional feature types.

How it Works:

  • The investigator prepares a single cell suspension from tissue or cells of interest
  • Core will count and verify cell viability of cell suspension
  • Core will then load cell suspension, gel beads, reagents, and partitioning oil onto a chip
  • The Chromium instrument automates generation of micro emulsions (GEMs) containing individual cells, functional gel beads containing unique barcoded primers for identifying each cell, and reagents for reverse transcription
  • Core personnel generate cDNA libraries from pooled GEMs to generate 10X Barcoded cDNA
  • Core personnel cleanup, amplify QC, and quantify the cDNA library
  • The core provides advice for UPMC Genomics Center, Genomics Research Core, or external provider to perform standard Illumina short-read sequencing
  • Core personnel aid investigator in data analysis including mapping sequences and clustering as desired
5’ Gene Expression
Measure the activity of immune cells and their targets.

 

What it Assays: The Chromium Single Cell Immune Profiling Solution is a comprehensive approach to simultaneously examine the cellular context of the adaptive immune response and immune repertoires of hundreds to tens of thousands of T and B cells in human or mouse on a cell-by-cell basis. With the addition of Feature Barcoding technology, you can also detect and analyze additional cellular readouts, such as cell surface markers and antigen specificity, to enhance immune cell phenotyping and study dynamic interactions between lymphocytes and target cells.

How it Works:

  • The investigator prepares a single cell suspension from tissue or cells of interest
  • Core will count and verify cell viability of cell suspension
  • Core will then load cell suspension, gel beads, reagents, and partitioning oil onto a chip
  • The Chromium instrument automates generation of micro emulsions (GEMs) containing individual cells, functional gel beads containing unique barcoded primers for identifying each cell, and reagents for reverse transcription
  • Core personnel generate cDNA libraries from pooled GEMs to generate 10X Barcoded cDNA
  • At the request of the Investigator, Core personnel will generate cDNA libraries for B and or T cell sequencing, providing the unique BCR and/or TCR from each B or T cell
  • Core personnel cleanup, amplify QC, and quantify the cDNA library
  • The core provides advice for UPMC Genomics Center, Genomics Research Core, or external provider to perform standard Illumina short-read sequencing
  • Core personnel aid investigator in data analysis including mapping sequences, clonotype analysis, and clustering as desired
ATAC Seq
Measure epigenetics by detecting open chromatin regions.

 

What it Assays: The Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin) Solution accelerates the understanding of the regulatory landscape of the genome, thereby providing insights into cell variability. The chromatin profiling of tens of thousands of single cells in parallel allows researchers to see how chromatin compaction and DNA-binding proteins regulate gene expression at high resolution.

How it Works:

  • The investigator prepares a single cell suspension from tissue or cells of interest
  • Core will count and verify cell viability of cell suspension
  • Single nuclei are isolated from cell suspension before being transposed
  • Core will then load pooled transposed nuclei, gel beads, reagents, and partitioning oil onto a chip
  • The Chromium instrument automates generation of micro emulsions (GEMs) containing single nuclei, functional gel beads containing unique barcoded primers for identifying each cell, and reagents for reverse transcription
  • Core personnel generate cDNA libraries from pooled GEMs to generate 10X Barcoded DNA fragments
  • Core personnel cleanup, amplify QC, and quantify the DNA fragment library
  • The core provides advice for UPMC Genomics Center, Genomics Research Core, or external provider to perform standard Illumina short-read sequencing
  • Core personnel aid investigator in interrogating open chromatin profiles and data analysis including mapping sequences and clustering as desired

Multiome ATAC and Gene Expression
Obtain transcriptome and open chromatin information from the same cell. Gene expression in this system is detected in nuclei, i.e., single nuclear RNA-seq and thus the resulting transcriptomes will look different from single cell RNA-sequencing studies.

What it Assays: Simultaneously profile gene expression and open chromatin from the same cell with Chromium Single Cell Multiome ATAC + Gene Expression. Multiply your power of discovery to characterize cell types and states, and uncover gene regulatory programs.

How it Works:

  • The investigator prepares a single nuclei suspension from tissue or cells of interest. For cells from tissues, this can be accomplished by detergent lysis of cell membranes after preparation of a single cell preparation or detergent lysis can be carried out directly on tissues or cell suspensions   
  • Core personnel verify successful cell lysis for the nuclear suspension and count nuclei
  • Core personnel will transpose cell nuclei, creating DNA fragments from open DNA (ATAC)
  • Core personnel will then load pooled transposed nuclei, gel beads, reagents, and partitioning oil onto a 10X Genomics partitioning chip
  • The Chromium instrument will generate micro emulsions (GEMs), containing single nuclei, functional gel beads containing unique barcoded primers for identifying each nucleus, and reagents for reverse transcription. This step will generate bar-coded cDNAs from the polyA nuclear mRNAs, and barcoded ATAC-seq, DNA fragments
  • Core personnel cleanup, amplify QC, and quantify the gene expression and ATAC/DNA fragment libraries
  • The core provides advice for UPMC Genomics Center, Genomics Research Core, or external provider to perform standard Illumina short-read sequencing
  • Core personnel aid investigator in interrogating open chromatin profiles and data analysis including mapping sequences and clustering as desired.

 

CITE Seq
RNA sequencing that provides quantitative and qualitative information of cell surface proteins along with gene expression. Barcoded antibodies bind to individual cells, attaching a marker to the mRNA sequence that carries over through the library preparation.

Barcoded antibodies bind to surface markers on individual cells (similar to flow cytometry), the barcode marker sequence carries over through the library preparation, permitting quantitative assessment of surface antibody binding to cells along with their transcriptome information. Hundreds of surface antigens can be assayed simultaneously.

Available antibodies and panels can be found at Biolegend: https://www.biolegend.com/en-us/totalseq

How it Works:

  • The investigator prepares a single cell suspension from tissue or cells of interest
  • The investigator labels cells with Cite-seq antibodies. Please inquire at the Core for available reagents. In many cases the Investigator will need to purchase these antibody-oligonucleotide reagents.
  • Core will count and verify cell viability of cell suspension
  • Core will then load cell suspensions(s), gel beads, reagents, and partitioning oil onto a chip
  • The Chromium instrument automates generation of micro emulsions (GEMs) containing single cells, functional gel beads containing unique barcoded primers for identifying each cell, and reagents for reverse transcription
  • Core personnel generate cDNA libraries from pooled GEMs to generate 10X Barcoded cDNAs and a second library for antibody-oligonucleotides
  • Core personnel cleanup, amplify QC, and quantify the libraries
  • The core provides advice for UPMC Genomics Center, Genomics Research Core, or external provider to perform standard Illumina short-read sequencing
  • Core personnel aid investigator in mapping sequences, clustering cell by transcriptomes and proteomic analysis as desired
Multiplexing samples/Cell Hashing
Utilizing barcoded antibody labeling to multiplex multiple cell preparations into one single reaction to considerably reduce the experimental cost and add experimental design flexibility: list of hashtags we provide:

 

Hashtags That the Core Provides:

TotalSeq-A is intended for use with the 3’ kits while TotalSeq-C is intended for use with the 5’ kits. Please note that while human cell hashing generally works well with all cells, currently murine cell hashing only works well with CD45+ cells (inquire at the core as we expect this issue to soon be resolved so that all murine cells can be multiplexed with hashtags).

TotalSeq™ anti-human Hashtag reagents are designed to label most human cells, using a combination of two clones that recognize CD298 and β2 microglobulin, respectively.

  • TotalSeq-A anti-human Hashtags 1 through 14
  • TotalSeq-C anti-human Hashtags 1 through 10

TotalSeq™ anti-mouse Hashtag reagents are a mixture of two monoclonal antibodies conjugated to the same oligonucleotide. The antibodies are specific against mouse CD45 and MHC class I (of a, b, d, j, k, s, and u haplotypes).

  • TotalSeq-A anti-mouse Hashtags 1 through 15
  • TotalSeq-C anti-mouse Hashtags 1 through 6

Not all mouse strains are recognized with the antibodies. Please make sure your samples are compatible.

Mouse strains recognized: 129/-; A/J; AKR/J; BALB/cAnN; BALB/cBy; BALB/CJ; BXSB/Mp; C3H/Bi; C3H/He; C3HeB/FeJ; C57BL/6; C57BL/10; C57BLR/cdj; C57L/J; C58/J; C.B-17; CBA/Ca; CBA/J; CBA/N; CE/J; DA/HuSn; GRS/J; HRS/J; I/LnJ; LP/J; MA/MyJ; MRL/Mp; NOD; NZB/-; PL/J; RF/J; SEC/-; SJL/J; ST/bJ; SWR/J.

BCR Library
Characterizing the BCR repertoire and linking BCR sequences to specific cell subsets. (compatible with all 5’ gene expression libraries)

 

  • Pair heavy and light chain immunoglobulin (Ig) sequences from individual B cells with full isotype resolution
  • Assemble and annotate full-length V(D)J gene sequences
TCR Library
Characterizing the TCR repertoire and linking TCR sequences to specific cell subsets. (compatible with all 5’ gene expression libraries)

 

  • Pair α and β chain TCR sequences from individual T cells
  • Assemble and annotate full-length V(D)J gene sequences
  • Link full-length, paired TCR α and β chain sequences with TCR-pMHC specificity
Bioinformatics
We offer sequencing and analysis for your single cell data, including sequence mapping, clustering, violin plots, dot plots, BCR and TCR clonotype analysis, proteomics analysis (Cite-seq) and open chromatin analysis (scATAC-seq). For more information, please contact us here.

 

Contact Us

Contact us for a free consultation regarding your next single cell experiment.