Periodic Reporting for period 3 - CellKarma (Dissecting the regulatory logic of cell fate reprogramming through integrative and single cell genomics)
Período documentado: 2021-03-01 hasta 2022-08-31
In summary, I developed all points described in each project task and also amplified the range of outcomes with novel and more efficient strategies. Some of such choices have also been undertaken as a natural adaptation to the ongoing scientific field changes and demands.
Furthermore, from a more technically-related point of view, I originally planned to use single-cell approaches to obtain transcriptional information on a maximum of 3.000 genes in individual cells. My team set up a successful protocol that allowed the profiling at high resolution of up to 6.000 genes per cell, thus further increasing the information on ongoing molecular dynamics.
In addition, we validated an approach to obtain transcriptomic and epigenomic data from the same experiment, combining the single-cell RNAseq (scRNAseq) with scATACseq approach.
In Aim 4, I originally proposed to validate candidates by modulating their expression in a primary reprogramming model but the implementation of the microfluidic approach allowed the discovery of novel candidates and the simultaneous direct validation, thus accelerating the overall project performance. Therefore, I decided to use the TERA (Transcription Factor Epigenetic Remodeling Activity) score to carry on a de novo discovery on a list of 300 TFs, ranked on their ability to act on cellular plasticity. On such TFs, we already performed a comprehensive dosage profiling using RNAseq, ATACseq, morphology assay (immunofluorescence-based) and CUT&RUN (cleavage under targets and release using nuclease) sequencing approach to study each TF binding to DNA.