Single Cell Sequencing

Question 1

An advantage of single cell RNA sequencing over Bulk RNA-seq?

Answer 1

Can identify cell specific changes.
Cell specific not tissue specific.

Question 2

How are droplets formed during drop-seq? What do the droplets contain?

Answer 2

Microfluidics device
Combination of aqueous solutions and oil to create small droplet containing cell and barcoded bead.

Question 3

What prcoess leads to barcodes binding to mRNA transcripts within droplet?

Answer 3

Lysis occurs in droplet, releasing all mRNA from cell.
mRNA is reverse transcribed into cDNA.
Universal primers attached to the DNA barcodes fascilitate binding to mRNA. .

Question 4

What is the difference between Unique Molecular Identifiers (UMI) and barcodes?

Answer 4

The cell barcode distinguishes between cells, and the UMI distinguishes between molecules.

Question 5

What is the composition of a primer in a droplet?

Answer 5

• complementary sequence binding to target mRNA
• UMI
• barcode

Question 6

Does UMI target specific molecules?

Answer 6

No.
UMI is random sequence that is assigned to a molecule upon binding.

Question 7

Why does mRNA undergo reverse transcription into cDNA after cell lysis?

Answer 7

• higher stability
• amplification (must be double stranded to go through PCR)
• sequencing (many require DNA as the input material)

Question 8

What do the rows and columns of the count matrix represent?

Answer 8

Rows: each row represents a specific gene.
Columns: each column represents an individual cell (droplet).