Periodic Reporting for period 2 - SC-EpiCode (Decoding the Epigenomic Regulatory Code by the Use of Single Cell Technologies)
Reporting period: 2019-04-01 to 2020-09-30
For Aim 2 and 3, we have generated mouse ES cell lines perturbed in multiple chromatin regulator genes rendering the cells viable and able to differentiate. This was done by cloning of a lentiviral plasmid bearing tet-on Cas9 fused to a GFP reporter (TRE-Cas9-GFP-tetON), it yielded a cell line expressing Cas9 upon Doxycycline activation. By analyzing the resulting genetic background of the perturbed cell lines, we verified the effectiveness of the perturbations, while the robustness of perturbations needed to be refined.
Finally, we just published a paper ""Transcription Factor Binding in Embryonic Stem Cells Is Constrained by DNA Sequence Repeat Symmetry"" in Biophysical Journal for which we reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. Using an in vivo reporter assay, we show that gene expression in embryonic stem cells can be positively modulated by the presence of genomic and computationally designed DNA oligonucleotides containing identified nonconsensus-repetitive sequence elements. Our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry."
Meanwhile, we constated that the available technologies for single-cell sequencing nowadays are either microfluidics-based that allow routine profiling of thousands of single cells in an experiment but sequence only the 3’ end and cannot recover splicing patterns or sequence variants, or well plate-based that allow a better sensitivity per cell and enable sequencing of the full transcripts with an individual amplification strategy but suffer from high-cost, labour-intensity and small-scale. Therefore, we are developing a novel 3D clone-based full-length RNA-Seq profiling technology, which will be the first time a full-length RNA-seq is done in drop-based microfluidics. We modify the conventional DGE (Differential Gene Expression) 3’-sequencing into full-length RNA sequencing in droplets by using Not So Random (NSR) primers, specific 408 hexamers targeted to human mRNA but depleted from rRNA and tRNA will be constructed within our barcoded hydrogels using a dedicated robotic liquid handler. This work is currently under revision for ERC Proof of Concept Pilot Lump Sum Grant 2020.