and ERCC RNA Spike-in control Control IL2 treated Control IL2 - - PowerPoint PPT Presentation

Mapped RNA- seq reads data for SRIL2+ and SRIL2- Mapped RNA- seq reads data for SENIL2+ and SENIL2- Mapped RNA- seq reads data for REJIL2+ and REJIL2-

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Self-renewing hADSC Senescent hADSC Rejuvenated hADSC Control IL2 treated Control IL2 treated Control IL2 treated

Cell culture IL2 treatment and ERCC RNA Spike-in control RNA-seq and reads mapping

IL2 ERCC mix ERCC mix IL2 ERCC mix ERCC mix IL2 ERCC mix ERCC mix

SR SEN REJ

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ERCC Report

ERCC_Analysis plugin

Allows for

• Determining dynamic range
• Assessing lower limit of detection
• Detecting fold-change response

ERCC Analysis

• The ERCC_Analysis plugin

is a software package that comes with the Ion Proton machine.

• The plugin is used in RNA-

seq experiments with ERCC RNA Spike-In Control Mixes

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SRIL2+ SRIL2- SENIL2+ SENIL2- REJIL2+ REJIL2- SR+ SR- SEN+ SEN- REJ+ REJ-

compare compare compare

• Genes up-regulated in SR upon IL2

treatment

• Genes down-regulated in SR upon IL2

treatment

• Genes up-regulated in SEN upon IL2

treatment

• Genes down-regulated in SEN upon

IL2 treatment

• Genes up-regulated in REJ upon IL2

treatment

• Genes down-regulated in REJ upon IL2

treatment

Differential expression(DE) analysis

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SR+ SR- SEN+ REJ+ REJ- SEN-

Comparing DE gene sets

Up-regulated genes upon IL2 treatment Down-regulated genes upon IL2 treatment

• After obtaining DE genes for each cell types, we can look at the genes

which are unique to specific cell type or shared by two or more cell types.

• All results for the DE genes will be put in a single excel file in which the

genes can be sort and filtered.

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SR+ SEN+ REJ+ 1 2

Up-regulated genes upon IL2 treatment

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• In this example, up-regulated genes in SR, SEN and REJ are compared.
• This will results in seven subsets of genes among which subset No.1 is shared

by all three cell types, subset No.2, No.3 and No.4 are shared by two of the three cell types and subset No.5, No.6 and No.7 are unique to SR, SEN and REJ, respectively.

Example of up-regulated genes

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Example of excel tables for up-regulated genes

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Gene symbol Level of differential expression A “1” in each column represents that the DE gene belongs to this set while a “0” means not. When there is only

• ne “1” the three

columns(C,D,E), the gene uniquely belongs to one set. When there are more than one “1” in the three columns, the gene is shared

• In this excel file, it is easy to sort genes by shared or unique status.
• It is also easy to use filter in Excel to find out genes unique to (or shared by) particular set(s) based on

“1” and “0” values in columns C, D and E.

• For example, “Gene3” has “1”s in column C, D and E, this shows that “Gene3” belongs to subset No.1,

which contains genes shared by all three cell types. “Gene6” has “0”s in column C and D, and “1” in column E, this shows that “Gene6” is uniquely up-regulated in REJ cells.

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Start Week 1 Week 2 Week 3 Week 4

• Agreement on
• verall

analytical design

• Confirmed

data file location and description

• Completed

data transfer

• Visualization

for pathway and functional analysis

• Quantify

expression

• Data

normalization

• Differential

expression analysis

• Compare

shared and unique DE genes among three conditions

• Functional

annotation for individual shared or unique genes

• f interest
• Functional

enrichment analysis for publicly available gene sets

• Functional

enrichment analysis for customized gene sets Deliverables

Six lists of DE genes including up- and down- regulated ones Lists of shared and/or unique DE genes depending on questions to address Lists of statistically significant pathways and/or functional sets with p-values Visualized gene-gene interaction in terms of networks and/or diagrams