Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues.
BACKGROUND
The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.


RESULTS
We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.


CONCLUSIONS
Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.
Ammar
A
Zaghlool
Adam
A
Ameur
Linnea
L
Nyberg
Jonatan
J
Halvardson
Manfred
M
Grabherr
Lucia
L
Cavelier
Department of Immunology, Genetics and Pathology, Rudbeck Laboratory and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. lars.feuk@igp.uu.se.
Lars
L
Feuk
D002460:Cell Line; D003593:Cytoplasm / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007438:Introns; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
D012333:RNA, Messenger