Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

Abstract

BackgroundPlatelets 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 MethodsWe 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.ResultsConsistent 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.ConclusionThe 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.

Circular RNA enrichment in platelets is a signature of transcriptome degradation

“…10,11 Collectively, our results indicate that translation in platelets occurs against the backdrop of a rapidly degrading transcriptome, that platelets will differ in their translational potential in an agedependent manner, and that RNAseq analyses targeted toward intact species that are capped 15,18 or polysome bound will be required to analyze translational potential in these cells effectively.…”

“…In silico analyses were performed on RNAseq data generated during this study and on 36 publicly available datasets: 24 ribosomal RNA depleted total RNA samples sequenced on the Illumina platform, 11,23,27,36,37 2 polyA1 selected samples sequenced on the Ilumina platform, 11,38 and 10 nonribosomal RNA depleted total RNA samples sequenced on the SOLiD platform. 15 For details, see supplemental Methods and supplemental Table S1 available on the Blood Web site.…”

“…We first identified 3 publicly available RNAseq datasets from human platelets, 9,11,15 one of which had a sequence read length long enough for the identification of back-splice junctions within circRNAs and included samples processed to retain unpolyadenylated RNAs (Array express ID E-MTAB-1846; 100-bp Illumina ribosome-depleted total RNAseq from 3 individuals, and polyA 1 RNAseq from 1 individual…”

“…6,7 Consistent with these observations, recent RNAseq analyses have defined a complex platelet transcriptome. [8][9][10][11] However, some transcripts believed to contribute to platelet function are present at very low levels in RNAseq data, 11 and attempts to integrate transcriptome and proteome data have given conflicting results. [12][13][14][15] In the absence of nuclear transcription, RNAs within platelets are assumed to degrade over time.…”

“…9,11,15,17,18 Transcripts with rearranged exon order are now known to be common in eukaryotic organisms and are characterized by backsplice exon junctions inconsistent with the underlying genomic DNA. [19][20][21][22][23][24] Evidence for linear and polyadenylated structures has been presented, 20,25,26 but it is now clear that most are circular (circRNAs) and cytoplasmic.…”