title: Two methods for full-length RNA sequencing for low quantities of cells and single cells.
abstract: The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA. xinghua.pan@yale.edu
authors.forename: Xinghua
authors.initials: X
authors.lastname: Pan
authors.forename: Russell E
authors.initials: RE
authors.lastname: Durrett
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhu
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Yumei
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Zi
authors.forename: Sadie L
authors.initials: SL
authors.lastname: Marjani
authors.forename: Ghia
authors.initials: G
authors.lastname: Euskirchen
authors.forename: Chao
authors.initials: C
authors.lastname: Ma
authors.forename: Robert H
authors.initials: RH
authors.lastname: Lamotte
authors.forename: In-Hyun
authors.initials: IH
authors.lastname: Park
authors.forename: Michael P
authors.initials: MP
authors.lastname: Snyder
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Mason
authors.forename: Sherman M
authors.initials: SM
authors.lastname: Weissman
mesh_terms: D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D020014:K562 Cells; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger