10x Genomics single cell library preps

For new users, a consultation is required prior to submitting an order. Please contact Dr. Katia Sol-Church (ks5uq@virginia.edu) to set up a meeting time.  All consultations are free.

After you have discussed your project, please place an order in iLab using our ’10xGenomics library prep and sequencing NGS’ project request. A form will guide you through the order placement process. If you have any questions about placing your order, please contact  Alyson Prorock (ajp7x@virginia.edu) for assistance.

Remember, we encourage the use of miSeq QC run to check the number of targeted single cells before proceeding to full scale sequencing on the NS2K.

This is the most important step in your experiment.  One needs to make sure that the cells loaded into the chromium system are viable and single cells. Cell aggregates or debris can increase the risk of clogs on a microfluidic chip.  Likewise, for nuclei prep, make sure they are intact and that they contain less than 5% live cells.  Optimizing the tissue dissociation protocol for your particular sample type is paramount to getting high quality single cells or nuclei devoid of free floating RNAs that can contribute to the background noise.

Protocols are available on the 10X Genomics site that are cell/tissue specific, however I recommend each lab  to optimize their cell/ nuclei prep in advance of the chromium appointment.

10X Genomics has optimized several protocol that are commonly used in the core to prepare  library preps.  These include  indexed 3′ or 5′ gene expression  libraries, T or B cell immune profiling,  cell surface expression (CITE-seq and Cell Hashing), ATAC-seq and single nuclei multiOmics (ATAC-seq plus Gene expression).

The Chromium Single Cell 5′ Library Construction Kit and  V(D)J Enrichment Kit offer comprehensive, scalable solutions for measuring gene expression and immune repertoire information from the same cell. Profile full-length (5’ UTR to constant region), paired T-cell receptor (TCR), or B-cell immunoglobulin (Ig) transcripts from 100-10,000 individual cells per sample. A pool of ~750,000 barcodes are sampled separately to index each cell’s transcriptome. It does so by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions.

This protocol can be used to generate an enriched T-cell library and/or an enriched B-cell library, and/or a 5’ Gene Expression library from amplified cDNA from the same cells.

The Chromium Single Cell Feature Barcode technology offers a comprehensive, scalable approach to detect cell surface proteins along with the gene expression and immune repertoire information from the same single cell. This is accomplished by labeling cell surface proteins with antibodies conjugated to a Feature Barcode oligonucleotide, followed by direct capture of the Feature Barcode by the Gel Bead primer. A pool of ~750,000 barcodes are sampled separately to index each cell’s transcriptome and cell surface protein. It does so by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions.

Use this technique to measure both gene and cell surface protein expression in the same cell to identify protein isoforms, detect protein for low abundance transcripts, and further increase phenotypic specificity. This technology can also determine antigen specificity of single T cells with Feature Barcode peptide-MHC multimers to study the dynamic interactions between lymphocytes and antigens. It can also be used to multiplex more than one sample into a single library prep.

This service can be performed in conjunction with the 5′ Next GEM library prep or the VDJ immune profiling (B cell or T cell) library prep, but is not a stand alone service.

Download the 10x Genomics user guide here: Feature Barcode for Cell Surface Protein

Uses Chromium Single Cell 5′ Feature Barcode Library Kit (10x Genomics, 1000080)

The Chromium Single Cell ATAC Solution provides a comprehensive, scalable approach to determine the regulatory landscape of chromatin in hundreds to thousands of cells in a single sample. This is achieved by transposing nuclei in a bulk solution; then, using a microfluidic chip and the Chromium Controller, the nuclei are partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs). A pool of ~750,000 10x Barcodes is sampled to separately and uniquely index the transposed DNA of each individual nucleus. Libraries are generated and sequenced, and 10x Barcodes are used to associate individual reads back to the individual partitions, and thereby, to each individual nucleus.

Download the 10x Genomics user guide here: ATAC-seq user guide

Uses Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10x Genomics, 1000175/1000176) and
Chromium Next GEM Chip H Single Cell Kit (10x Genomics, 1000161/1000162)