Single-Cell Sequencing Analysis

A. 10× Genomics Chromium Single-Cell Analysis

The BIG Core provides 10× Genomics Chromium Controller-based, high-throughput, scalable single-cell/nucleus analysis, which encapsulates thousands of individual cells/nuclei in droplets for lysis and the following enzymatic reactions from up to 8 samples in parallel. Generated by microfluidics and the Gel Bead-in-Emulsion (GEM) technology, the droplets contain adapters of cell/nucleus- and molecule-specific barcoding for single-cell/nucleus library preparation. The resulting pooled libraries are then sequenced on Illumina NextSeq 2000.

A.1   Single-Cell Gene Expression (scRNA-Seq, 3′ end or 5′ end)

Measure gene activity on a cell-by-cell basis and characterize cell populations, cell types, differential gene expression, and more.

A.2   Single-Cell ATAC (scATAC-Seq)

Measure epigenetics, genome-wide chromatin accessibility, on a cell-by-cell basis by detecting open chromatin regions.

A.3   Single-Cell Multiome (scATAC-Seq + scRNA-Seq)

Measure genome-wide gene expression and chromatin accessibility simultaneously from the same single cell, across thousands of cells.

A.4   Single-Cell Immune Profiling

Measure the activity of immune cells and their targets by simultaneous generation of full-length, paired V(D)J sequences, as well as gene expression, cell surface protein expression, and antigen specificity data at a single-cell level.

B. SMART-Seq Single-Cell Analysis

SMART (Switching Mechanism at 5′ end of RNA Template) technology relies on the template-switching activity of reverse transcriptase to enrich full-length cDNAs and to add defined PCR adapters directly to both ends of the first-strand cDNA. This ensures that the final cDNA libraries contain the entire length of the mRNA, from the 3′ to 5′ end, maintaining a true representation of the original mRNA transcripts. For scRNA-Seq using SMART technology, investigators are encouraged to sort individual cells into the wells of a 96-well plate containing lysis buffer and perform reverse transcription followed by PCR amplification at their own convenience. Investigators can DIY (do-it-yourself) with the published protocol or use the Takara SMART-Seq Single-Cell commercial kit; provide the BIG Core with the cDNA amplified or the NGS libraries constructed in 96-well plates. We will provide troubleshooting assistance, if needed, to ensure the successful completion of your project.