In situ Sequencing Platforms for Investigating Cellular Variations and Lineage
Je H. Lee
Cold Spring Harbor Laboratory, NY, USA
Despite advances in next-generation sequencing (NGS) and modern imaging techniques, fundamental barriers limit the scalability and the resolution of de novo transcriptome reconstruction in space. To address this, our lab is developing a suit of sequencing, imaging, and computational approaches toward reconstructing the transcriptome in 3D space from thousands of single cells and embryos that are imaged in parallel (Fluorescent in situ RNA sequencing; FISSEQ).
Cell-cell interactions are critical modifiers of cell signaling and gene transcription in vivo, including short-range (direct contact), medium-range (growth factors), and long-range (mechanotransduction) communications. We hypothesize that cellular decision-making might depend on their ability to integrate such interactions using lineage-specific transcriptional programs. By analyzing the transcriptome changes in situ, we hope to reveal how cells use their 'geolocation' information to determine cell fate in development.
Over the past year, we have implemented an in situ sequencing platform capable of high-throughput 3D imaging. We can now image and sequence 104 cells containing stochastically sampled transcriptome amplicons in space for generating a tomographic representation of subcellular or intra-colony RNA expression patterns genome-wide. We believe that our method might allow for the investigation of topological constraints that determine transcriptional competency in development, especially when combined with micropatterning, genetic perturbations, and live cell imaging. In addition, we will present unpublished data on our new in situ sequencing chemistry directly on the RNA template for simpler approaches to targeted RNA sequencing, cell labeling, and Drosophila cell lineage reconstruction in 3D space.
Funding support: NIGMS R35 MIRA, STARR, V Foundation, HSFP, Pershing, CSHL