SR SEN REJ Cell culture Self-renewing hADSC Senescent hADSC Rejuvenated hADSC ERCC mix ERCC mix ERCC mix ERCC mix ERCC mix ERCC mix IL2 treatment IL2 IL2 IL2 and ERCC RNA Spike-in control Control IL2 treated Control IL2 treated Control IL2 treated Mapped RNA- Mapped RNA- Mapped RNA- RNA-seq and seq reads data seq reads data seq reads data for SR IL2+ and for SEN IL2+ and for REJ IL2+ and reads mapping SR IL2- SEN IL2- REJ IL2- 1
ERCC Analysis Allows for • Determining dynamic range ERCC_Analysis ERCC Report plugin • Assessing lower limit of detection • Detecting fold-change response • 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 2
Differential expression(DE) analysis • Genes up-regulated in SR upon IL2 SR IL2+ SR + treatment compare • Genes down-regulated in SR upon IL2 SR IL2- SR - treatment • Genes up-regulated in SEN upon IL2 SEN + SEN IL2+ treatment compare • Genes down-regulated in SEN upon SEN IL2- SEN - IL2 treatment • Genes up-regulated in REJ upon IL2 REJ + REJ IL2+ treatment compare • Genes down-regulated in REJ upon IL2 REJ IL2- REJ - treatment 3
Comparing DE gene sets Up-regulated genes upon IL2 treatment Down-regulated genes upon IL2 treatment SEN + SEN - SR - SR + REJ + REJ - • 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. 4
Example of up-regulated genes Up-regulated genes upon IL2 treatment SEN + SR + 3 6 5 4 1 2 7 REJ + • 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. 5
Example of excel tables for up-regulated genes When there is only A “1” in each one “1” the three column columns(C,D,E), the Level of represents gene uniquely differential that the DE belongs to one set. expression gene belongs When there are to this set more than one “1” while a “0” in the three means not. Gene symbol columns, the gene is shared 6 3 1 5 2 7 4 • 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. 6
Start Week 1 Week 2 Week 3 Week 4 • • • • • Agreement on Quantify Compare Functional Visualization overall expression shared and enrichment for pathway analytical unique DE analysis for and functional • design Data genes among publicly analysis normalization three available gene • Confirmed conditions sets • data file Differential • • location and expression Functional Functional description analysis annotation for enrichment individual analysis for • Completed shared or customized data transfer unique genes gene sets of interest Lists of Visualized Lists of shared Six lists of DE statistically gene-gene and/or unique genes significant interaction in DE genes including up- pathways terms of Deliverables depending on and down- and/or networks questions to regulated ones functional sets and/or address with p-values diagrams 7
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