Single Cell Transcriptome Amplification with MALBAC

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.Chemistry and Chemical Biolog

Single cell transcriptome amplification with MALBAC.

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

Gene expression profiles of 7.0dpc mouse embryo stem cells from 3 different germ layers.

<p>MALBAC-RNA distinguishes single cells from different germ layers of a post-implantation mouse embryo (7.0dpc). A total of 12 single cells were isolated from a 7.0dpc mouse embryo, among which 3 were from the ectoderm, 5 from the mesoderm, and 4 from the visceral endoderm. (A) Principle component analysis of transcriptomes clearly separates the 12 single cells into three clusters, each representing one germ layer. (B) Top: Hierarchical clustering of transcriptomes classifies the 12 single cells into three non-overlapping sub-trees representing the three germ layers. Bottom: Known marker genes of the three germ layers exhibit strong layer-specific patterns of expression, although some show significant cell-to-cell variation within a layer. Principle component analysis and hierarchical clustering were based on the ranking of each gene’s FPKM among all cells.</p

Single-cell MALBAC-RNA amplification diagram.

<p>After reverse transcription, primers with 7 random nucleotides at the 3’ end are annealed to the cDNA template at 4°C, then extended by DNA polymerase with strand displacement activity as temperature is increased. Amplicons are then melted off the original template after DNA extension, and looped at 58°C to protect themselves from being further amplified thanks to their 5’ ends being complementary to their 3’ ends. This MALBAC-RNA step includes a total of 10 cycles of quasilinear amplification, followed by another 19 cycles of PCR.</p

Technical reproducibility of MALBAC-RNA amplification.

<p>(A) Mean expression level measured in across two technical replicates and nine SW480 single cells for synthetic spike-ins of a particular concentration. Error bars represent standard errors. (B) Scatter plot of two technical replicates exhibits a high correlation coefficient (R = 0.995). To prepare technical replicates, single-cell amount of RNA was aliquoted from 100 cells after cell membrane lysis and they should only differ by Poisson fluctuations in molecular counts. (C) Probability of detecting a transcript in one technical replicate as a function of its expression level in the other replicate. (C) Probability that the expression level of a transcript in one replicate will differ by at least 10-fold from the measurement in the other replicate.</p

Gene expression heat map of EMT-related genes.

<p>Genes related to epithelial-mesenchymal transition (EMT) are differentially expressed across the three germ layers. Among them, FGF10 and Snai1 are significantly overexpressed in the mesoderm, whereas E-cadherin and Sox3 are depleted. At the same time, Eomes and Mesp1 are highly expressed in the mesoderm, although Mesp2 is not significantly expressed. Other EMT-related genes, including CDH2, Wnt5a, Wnt3, Hmga2, Smad1, and Fgf10, are also enriched in the mesoderm, which confirms the cellular transitions during gastrulation.</p