Complete Transcriptome RNA-Seq.
RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifications by fixation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNA-Seq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, we use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Our protocol utilizes duplex-specific nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profiling from low quantity input. We employ the Illumina sequencing platform, but this method is described in sufficient detail to adapt to other platforms.
Medical Sciences Program, Indiana University School of Medicine, 1001 East 3rd Street, Bloomington, IN, 47405, USA. millerdf@indiana.edu.
David F B
DF
Miller
Department of Internal Medicine, OSUCCC-Illumina Core, 460 West 12th Street, Columbus, OH, 43210, USA.
Pearlly
P
Yan
Medical Sciences Program, Indiana University School of Medicine, 1001 East 3rd Street, Bloomington, IN, 47405, USA.
Fang
F
Fang
Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, 47405, USA.
Aaron
A
Buechlein
Department of Internal Medicine, OSUCCC-Illumina Core, 460 West 12th Street, Columbus, OH, 43210, USA.
Karl
K
Kroll
Department of Internal Medicine, OSUCCC-Illumina Core, 460 West 12th Street, Columbus, OH, 43210, USA.
David
D
Frankhouser
Department of Internal Medicine, OSUCCC-Illumina Core, 460 West 12th Street, Columbus, OH, 43210, USA.
Cameron
C
Stump
Department of Internal Medicine, OSUCCC-Illumina Core, 460 West 12th Street, Columbus, OH, 43210, USA.
Paige
P
Stump
Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, 47405, USA.
James B
JB
Ford
Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, 47405, USA.
Haixu
H
Tang
Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, 47405, USA.
Scott
S
Michaels
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, 47405, USA.
Daniela
D
Matei
Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229, USA.
Tim H
TH
Huang
University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Jeremy
J
Chien
Center for Computation Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
Yunlong
Y
Liu
Indiana University Center for Genomics and Bioinformatics, Bloomington, IN, 47405, USA.
Douglas B
DB
Rusch
Medical Sciences Program, Indiana University School of Medicine, 1001 East 3rd Street, Bloomington, IN, 47405, USA. knephew@indiana.edu.|Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA. knephew@indiana.edu.|Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA. knephew@indiana.edu.
Kenneth P
KP
Nephew
D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D015345:Oligonucleotide Probes / Q000737:chemistry; D059367:RNA Cleavage; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012260:Ribonucleases / Q000737:chemistry; D017423:Sequence Analysis, RNA; D059467:Transcriptome
D015345:Oligonucleotide Probes; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012260:Ribonucleases
Duplex-specific Nuclease; Gene Expression; RNA-Seq; Sequencing; Transcriptome