Tracing the derivation of embryonic stem cells from the inner cell mass by single cell RNA-seq analysis

Here we modified a single cell whole transcriptome amplification method to make it capable of amplifying cDNAs as long as 3kb efficiently and unbiasedly. We combined this modified single cell cDNA amplification method with Applied Biosystems next generation sequencing SOLiD™ System to set up a single cell whole transcriptome assay. The modified amplification strategy allows us to amplify full-length cDNAs for most of the expressed genes. We show that it is feasible to get digital gene expression profiles at single cell resolution. This allows us to ask fundamental biological questions that could not be addressed previously, especially in the embyronic stem cell studues , and to understand transcriptome complexity at the resolution of a single cell.Four-cell stage embryos were recovered from MF1 females mated with MF1 male mice (Nagy et al. 2003). The zona pellucida was removed by treatment with acidic tyrode solution. The individual blastomeres were separated by gentle pipeting using a glass capillary. Mature oocytes were isolated from Dicer knockout [Dicer-/Flox, Zp3-Cre] and Ago2 knockout [Ago2-/Flox, Zp3-Cre] female mice (14,17). Cumulus cells were removed from oocytes by treatment with hyaluronidase. All the ‘mature oocytes’ mentioned in the text are ovulated mature oocytes.