Stem cell transcriptome profiling via massive-scale mRNA sequencing.
We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.
Cloonan
Nicole
N
Expression Genomics Laboratory, Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St. Lucia, Queensland, 4072, Australia.
Forrest
Alistair R R
AR
Kolle
Gabriel
G
Gardiner
Brooke B A
BB
Faulkner
Geoffrey J
GJ
Brown
Mellissa K
MK
Taylor
Darrin F
DF
Steptoe
Anita L
AL
Wani
Shivangi
S
Bethel
Graeme
G
Robertson
Alan J
AJ
Perkins
Andrew C
AC
Bruce
Stephen J
SJ
Lee
Clarence C
CC
Ranade
Swati S
SS
Peckham
Heather E
HE
Manning
Jonathan M
JM
McKernan
Kevin J
KJ
Grimmond
Sean M
SM
D000818:Animals; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods* / Q000706:statistics & numerical data; D015723:Gene Library; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics*; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*; D015398:Signal Transduction
D012333:RNA, Messenger