Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA

PLoS One. 2013 Dec 11;8(12):e81809. doi: 10.1371/journal.pone.0081809. eCollection 2013.

Abstract

Background: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA.

Materials and methods: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts.

Results: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq.

Conclusion: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and tissue types. The in vivo functional significance of the non-mitochondrial transcripts remains to be shown.

MeSH terms

  • Base Sequence
  • Blood Platelets / chemistry*
  • Blood Platelets / metabolism
  • Cytoskeleton / chemistry
  • Cytoskeleton / metabolism
  • Female
  • Gene Expression
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Leukocyte Common Antigens / genetics
  • Leukocyte Common Antigens / metabolism
  • Male
  • Mitochondria / chemistry*
  • Mitochondria / genetics
  • Molecular Sequence Data
  • Poly A / chemistry*
  • Poly A / genetics
  • RNA / chemistry*
  • RNA / genetics
  • RNA, Messenger / chemistry*
  • RNA, Messenger / classification
  • RNA, Messenger / genetics
  • RNA, Mitochondrial
  • RNA, Ribosomal
  • Sequence Alignment
  • Signal Transduction
  • Transcriptome*

Substances

  • RNA, Messenger
  • RNA, Mitochondrial
  • RNA, Ribosomal
  • Poly A
  • RNA
  • Leukocyte Common Antigens
  • PTPRC protein, human

Grant support

The County Council of Östergötland (A.K., J.J. and A.O.) and the Scientific Research Council (T.L., project VR 521-2012-2729) supported this project. Sequencing was performed by the SNP&SEQ Technology Platform, Science for Life Laboratory at Uppsala University, a national infrastructure supported by the Swedish Research Council (VR-RFI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.