I am a beginner in the programming area and I would like to ask for your help. I want to perform the deferential expression analysis using the DESeq2 package. I want to compare the expression of smallRNAs in three treated samples vs three control samples. I have the results from smallRNA-seq in txt files each for every sample, formated like this: sRNA_sequence raw_count TTTGGATTGAAGGGAGCTCTA 59493 Could you tell me how to prepare the file (dds) for the DESeq2 analysis?

Probably the best way is with tximport https://bioconductor.org/packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html

Look at the various ways to use it, and see which best fits the data format you have.

If all else fails, you can make your data into one matrix with samples as columns and genes as rows, DESeq can take that, but using tximport is preferable.