Single Cell/Nucleus sequencing

Question 1

What can be analysed using Bulk sequencing

Answer 1

Provides the average gene expression profile for a population of cells. The number of transcripts indicates how active a gene is. This can be an issue since a tissue is complex

Question 2

What are methods to isolate cells

Answer 2

1. FACS (Fluorescence-activated cell sorting). Using specific Ab’s to identify cells and then you can isolate a certain population of cells
2. MACS (Magnetic cell sorting).
   Using magnetic beads which is milder to the cells compared to FACS

Question 3

Which technique can be used instead of Bulk Sequencing to get information for specific cells

Answer 3

RNA-sequencing (Single cell)

Question 4

How can a sequence be labeled while wanting to sort sequences

Answer 4

Barcoding

Question 5

Name a barcode technique and its principle

Answer 5

Droplet based barcoding with the use of Beats. Any beat has a specific barcode attached to the beat, while will be attached to the cell after transcriping the sequence into the cell. Adding oil to cancel a waterdroplet so you have 1 droplet of oil and within that oil you will have a beat and a cell.

Question 6

What is a UMI which is also attached to the beat

Answer 6

Unique Molecular Identifier. After sequencing, the UMI is used to distinguish sequenced reads that originate from unique mRNA molecules VS PCR products

Question 7

Name some limitations regarding Single Cell RNA-seq

Answer 7

• Cell chanes and loss during cell sampling (ex. harsh detergents that are required to break up tissue can damage fragile cells leading to sample loss)
• Isolation procedures can lead to cell stress which can lead to changes in gene expression
• Single cell isolation is ONLY possible on fresh tissue and not archieved fixed material

Question 8

Name the advantages and disadvantages for using Single NUCLEUS RNA-seq

Answer 8

Advantages:
- Reducing biases in cell populations caused by cell dissociation
- Minimizing stress-induced transcriptional artifacts
- Reducing cell capture bias due to cell size, and the potential to capture nascent mRNA for temporal studies
- RNA can be taken from frozen cells/tissues

Disadvantages:
- Laborious nucleus preparation protocol
- Less RNA to start with

Question 9

Which RNA sequence technique will you use?

Case 1: Podocyte changes in congenital nephrotic syndrome patient –> germline mutation in the NPHS2 (podocin) gene

Answer 9

Since it’s congenital (born with it) germline mutation, all podocytes carry the mutation thus all podocytes has the same phenotype. BULK-SEQ could provide the answers. Sorting of podocytes from tissue is necessary. Compare data to data of healthy podocytes and observe differences

Question 10

Which RNA sequence technique will you use?

Case 2: Podocyte changes due to SARS-COV-2 infection (not congenital)

Answer 10

Subset of kidney cells including SOME podocytes are infected with SARS-COV-2. ONLY SCRNA-SEQ could provide the answers as only a small portion of the podocytes were infected. The podocytes need to be isolated. You don’t want to see the average gene expression since not every podocyte is infected