Presentation 3: RNA Sequencing

Question 1

How does Single cell RNA Seq work?

Answer 1

1) Isolate individual single cells from tissue sample
2) Sequence single cells by constructing cDNA libraries
3) Profile gene expression of single cells
4) Compare single cell data using bio-informatics

Question 2

How does Bulk RNA Seq differ from Single Cell RNA Seq?

Answer 2

Bulk: Average gene expression of all cells, and the true nature of tissue composition is hidden
Single Cell: Profile each individual cell’s gene expression and can get tissue composed of all sub-populations

Question 3

What is the disadvantage of Bulk RNA Seq?

Answer 3

Bulk RNA Seq can mask the gene expression profile of certain cells, cannot see gene expression in individual cells

Question 4

What is the advantage of Single cell RNA Seq?

Answer 4

Single Cell RNA seq shows gene expression in all cell types

Question 5

What was the goal of this study?

Answer 5

Provide new and individualistic biomarkers to improve diagnosis and treatment
Identify new candidate genes as biomarkers in lung cancer
Detect and profile differentially expressed genes in lung cancer

Question 6

What cell lines were chosen for this study and why?

Answer 6

A549, H460, Calu3, H1299 and all cells were identified as epithelial cells in lung cancer
Chosen to allow researchers the genetic profile, and high copy number plasmids, yielding more DNA per cell

Question 7

How was the Fluidigm used?

Answer 7

1) Obtained barcoded antibodies
2) Strain cells
3) Isolate antibody stained cells
4) Image
5) REAP- seq chemistry
Can only load each well with one cell, so used microscope to see.
The Fluidigm prepares the cDNA for the researchers

Question 8

How was the cDNA library prepared?

Answer 8

3’ end enriched cDNA library:
Preparation of cDNA diluents
Dual-barcoding
Bead purification

Question 9

How was the quality check performed?

Answer 9

Pool Libraries
Quantification using Bioanalyzer
Equimolar pooling
NGS using illumina sequencer

Question 10

How was the Illumina sequencer used?

Answer 10

1) Nucleic acid isolation
2) Library preparation
3) Sequencing: Clonal amplification or Sequencing reaction
4) Data analysis

Question 11

How was demultiplexing performed?

Answer 11

Barcode information to identify which sequence came from which sample
Read mapping for human genome
Detected differentially expressed genes

Question 12

How were single cell RNA sequencing libraries constructed?

Answer 12

400 cells from each cell line were indexed using dual barcoding
24, 424 genes mapped with greater than 2000 high quality reads per cell

Question 13

How were the quantification steps performed?

Answer 13

1) qRT-PCR
2) Gene set enrichment analysis
3) mRNA and microRNA levels
4) Available scRNA- seq data-sets
5) Western Blot Analysis

Question 14

How were DEGs detected?

Answer 14

Divided cells into clusters based on their gene expression profile
Cluster 1: Control because contained all cell lines, and in middle of the graph
To get DEGs compared clusters to cluster 1, and found 2,655 differentially expressed genes (DEG)

Question 15

What happened with the 2,655 newly found DEGs?

Answer 15

Divided into 6 gene sets, each set representing up or down regulation compared to the cluster comparison
Cluster 1 vs 5 had lower correlation

Question 16

How were the GSEA’s detected?

Answer 16

Used the cluster of cell lines, and took the DEGs for each cluster, the GO terms, the number of pathways, and number of signatures

Question 17

How was qRT-PCR used?

Answer 17

The 2,655 DEGS were ranked from highest to lowest change, and 40 DEGS per cluster were used
Validated the change in expression when compared to control A549 –> by validating the DEGS, positive correlation between single cell and bulk cell
Identified tumour associated antigens
Inverse expression found in up-regulation of C1 vs C4 comparison between scRNA (up) and bulk-RNA-seq (down)

Question 18

How was the validation of single cell RNA sequencing data performed by using publicly available data?

Answer 18

Compared the cluster analysis and CXCL2 found in diverse cell populations

Question 19

How was protein expression validated?

Answer 19

Western Blot and able to examine differences in expression at single cell level, biomarker potential, and association with epithelial mesenchymal transition