Single Cell Sequencing - Eric Chow (UCSF)

Learn why single cell sequencing is essential by comparing bulk versus single cell analysis using the fruit smoothie analogy. Discover how analyzing individual cells reveals distinct cell types and their transcriptional profiles that would be masked in bulk measurements.

Collapse high-dimensional gene expression data into two-dimensional plots using dimensionality reduction techniques. Analyze cell clustering patterns and use differential gene expression to classify distinct cell populations within the dataset.

Use a flow cytometer to sort individual cells into separate wells of a 96 or 384-well plate. Lyse each cell and perform reverse transcription using SMART-seq methodology with oligodT primers and template-switching oligos to generate full-length cDNA with PCR handles.

Amplify the cDNA through PCR to generate multiple copies, then use Nextera DNA library preparation to fragment the material and add sequencing adapters. Complete the library preparation process through additional PCR amplification.

Flow beads, cells, and oil through a microfluidic chip to create water-in-oil emulsion droplets. Most droplets will be empty, but a small fraction will contain both a single cell and a single bead with barcoded oligodT sequences.

Allow mRNA from encapsulated cells to be captured by the barcoded oligodT sequences on the beads. Retrieve beads from the droplets and perform reverse transcription with template-switching to generate cDNA, where all transcripts from a single cell share the same barcode identifier.