Hello

Please correct me if I am wrong as I am only getting a few significant genes after differential expression analysis

I have MSC cell lines (non-treated, IT treated and LPS treated) in 24 and 48 hours

I want to compare untreated MSC with IT and LPS so, my main table is this

> design
                                               condition time

First I look at 24 and 48 hours independently

For instance for 24 hours I done so

 > design
                                 X condition time
    1       CTRL_24_hrs_replicate1   control   24
    2       CTRL_24_hrs_replicate2   control   24
    3       CTRL_24_hrs_replicate3   control   24
    4  treatment_24_hrs_replicate1        t   24
    5  treatment_24_hrs_replicate2        t   24
    6  treatment_24_hrs_replicate3        t   24
    7       CTRL_48_hrs_replicate1   control   48
    8       CTRL_48_hrs_replicate2   control   48
    9       CTRL_48_hrs_replicate3   control   48
    10 treatment_48_hrs_replicate1        t   48
    11 treatment_48_hrs_replicate2        t   48
    12 treatment_48_hrs_replicate3        t   48
    > 

dds <- DESeqDataSetFromMatrix(countData=count, colData= design24, design=~condition, tidy = FALSE)

dds <- DESeq(dds)

resIT<- results(dds, contrast=c("condition", "IT", "control"))
 resLPS <- results(dds, contrast=c("condition", "LPS", "control"))
 resIT_LPS <- results(dds, contrast=c("condition", "IT", "LPS"))

I done the same for 48 hours

Looking at the results, I only see very few significant genes

head(resIT[order(resIT $padj),])

log2 fold change (MLE): condition IT vs control 
Wald test p-value: condition IT vs control 
DataFrame with 6 rows and 6 columns
                 baseMean log2FoldChange     lfcSE      stat      pvalue        padj
                <numeric>      <numeric> <numeric> <numeric>   <numeric>   <numeric>
hsa-miR-122-5p  2021.7353      -3.148194  0.519671  -6.05805 1.37781e-09 5.75699e-05
hsa-mir-122     2021.8461      -3.148297  0.519682  -6.05812 1.37722e-09 5.75699e-05
RNU11             38.5986      -2.882145  0.621620  -4.63651 3.54342e-06 9.87044e-02
hsa-mir-133a-1    13.6656      -2.571007  0.574599  -4.47444 7.66131e-06 1.28047e-01
hsa-miR-133a-3p   13.6656      -2.571007  0.574599  -4.47444 7.66131e-06 1.28047e-01
GNAI21           130.3099       0.993967  0.232581   4.27364 1.92304e-05 2.67838e-01

Please correct me where possibly I am doing something wrong, or how I can get more significant genes?

And, please how I could take time points into the design comparing my conditions without separation my data to 24 and 48 hours?

Anyway thanks a lot in advance

This is not the preferred way to do this. Instead, make a third column in coldata that joins time and condition, and use contrasts to indicate what subset to compare to what. But don't expect much to change.