Dataset Information

Single cell transcriptome amplification with MALBAC.

ABSTRACT: Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level.

SUBMITTER: Chapman AR

PROVIDER: S-EPMC4378937 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Single cell transcriptome amplification with MALBAC.

Chapman Alec R AR He Zi Z Lu Sijia S Yong Jun J Tan Longzhi L Tang Fuchou F Xie X Sunney XS

PloS one 20150330 3

Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mo ...[more]

PMID: 25822772

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Project description:Antigen-specific T cells play an essential role in immunoregulation and many diseases such as cancer. Characterizing the T cell receptor (TCR) sequences that encode T cell specificity is critical for elucidating the antigenic determinants of immunological diseases and designing therapeutic remedies. However, methods of obtaining single-cell TCR sequencing data are labor and cost intensive, typically requiring both cell sorting and full-length single-cell RNA-sequencing (scRNA-seq). New high-throughput 3' cell-barcoding scRNA-seq methods can simplify and scale this process; however, they do not routinely capture TCR sequences during library preparation and sequencing. While 5' cell-barcoding scRNA-seq methods can be used to examine TCR repertoire at single-cell resolution, doing so requires specialized reagents which cannot be applied to samples previously processed using 3' cell-barcoding methods.Here, we outline a method for sequencing TCRα and TCRβ transcripts from samples already processed using 3' cell-barcoding scRNA-seq platforms, ensuring TCR recovery at a single-cell resolution. In short, a fraction of the 3' barcoded whole transcriptome amplification (WTA) product typically used to generate a massively parallel 3' scRNA-seq library is enriched for TCR transcripts using biotinylated probes and further amplified using the same universal primer sequence from WTA. Primer extension using TCR V-region primers and targeted PCR amplification using a second universal primer result in a 3' barcoded single-cell CDR3-enriched library that can be sequenced with custom sequencing primers. Coupled with 3' scRNA-seq of the same WTA, this method enables simultaneous analysis of single-cell transcriptomes and TCR sequences which can help interpret inherent heterogeneity among antigen-specific T cells and salient disease biology. The method presented here can also be adapted readily to enrich and sequence other transcripts of interest from both 3' and 5' barcoded scRNA-seq WTA libraries.

Project description:BackgroundIn addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable.MethodsHere we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing.ResultsUsing microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power.ConclusionsOverall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.

Dataset Information

Single cell transcriptome amplification with MALBAC.

Publications

Single cell transcriptome amplification with MALBAC.

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