Transform and feed data into DESeq2 with DESeqDataSetFromMatrix
Error in DESeqDataSet(se, design = design, ignoreRank) : all variables
  in design formula must be columns in colData


I think that means just what it says...you don't have a "Groups" column in coldata.  Did you spotcheck your ColData data frame to make sure it looks the way it should?

&gt; cells &lt;- c("1","2","3")
&gt; groups &lt;- c("a", "b", "c")
&gt; test &lt;- data.frame(Cells &lt;- cells, Groups &lt;- groups)
&gt; test
  Cells....cells Groups....groups
1              1                a
2              2                b
3              3                c
&gt; colnames(test)
[1] "Cells....cells"   "Groups....groups"