Why my CD31+ endothelial cells RNAseq didn't express CD31?


Hi, I was doing RNAseq for CD31 selected human endothelial cells. Unfortunately, My cells didn't express CD31. I don't know why. The RNAseq method is SMART-Seq Stranded Kit (Takara).

I followed the general pipeline, Quality Control-Cutadapt-HISAT2-Stringtie. For the Stringtie, I used the reference file (hg19) to guide assembly.

For the gene abundance estimate of the Stringtie, the CD31(PECAM1) expression(FPKM) is 0.

Has anyone ever met this problem? What's the possible issue?







Are you sure there is no mismatch between annotation file and genome build?
I wouldn't rely on a single pipeline in this case, and specifically not the pipeline you are using.

  1. Try to look at the BAM files from the alignment, extract the genomic regions of CD31 from the BAM files and count reads using samtools and visualize the genomic region in IGV. If good coverage is detected, the problem is with stringtie, goto 5
    If still no or very few reads are found in this region:
  2. check genomic coordinates again,
  3. run alignment from scratch with a different (more sensitive) aligner (e.g. STAR),
  4. do a QC on the raw reads and the results, if sufficient:
  5. recount, eg. with HTseq, featureCount, if transcript counts needed try replacing step 3,5 by Salmon or Kallisto first.

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