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.RNA-seq | transcriptome amplification | full-length mRNA | single-cell analysis

From single-cell to cell-pool transcriptomes: Stochasticity in gene expression and RNA splicing

“…These are broadly referred to as ''single-cell RNA-seq'' (Tang et al 2009(Tang et al , 2010(Tang et al , 2011Ozsolak et al 2010;Islam et al 2011;Brouilette et al 2012;Cann et al 2012;Hashimshony et al 2012;Pan et al 2012;Qiu et al 2012;Ramsköld et al 2012). Despite these significant advances, there are substantial shortcomings in these methods, and a robust method for comprehensive and accurate measurement of the transcriptome of a single cell is not yet available.…”

Microfluidic single-cell whole-transcriptome sequencing

“…Recently, there has been a number of new approaches for low-quantity RNA-Seq (17)(18)(19)(20)(21), all with unique advantages and limitations. A notable approach, Smart-Seq, was developed to provide better coverage of full-length cDNAs for long mRNA molecules (19), and has undergone successive improvements since its inception (22,23), including a recent demonstration of absolute mRNA counting (24).…”

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

“…We used the amplified cDNA to make the template for qRT-PCR (Figure 1). There are several studies for full length RNA sequencing and measurement of RNA variability by using low quantities of cells and single cells 3,14,15 . To our knowledge, we diluted total RNA of hESCs (microgram amounts) down to nano-and pico-gram levels and applied our protocol to assess technical variability and detection of difference on low amounts of total RNA.…”