Purpose:

Correlating guide RNA identity to the transcriptome is the gold-standard in single cell PerturbSeq studies regardless of the scale, host organism or purpose of the screen. Here we present a novel innovation to co-interrogate both types of RNA molecules simultaneously using free space-scalable fluid reactions facilitated by magnetic, dual-oligo functionalized polymer particle induced compartmentalization called particle-templated instant partitions (PIPs). Because these reactions are completely fluid, they are fundamentally scalable from small discovery experiments to genome-wide studies without changing the molecular design or experimental design. However, in previous design approaches, the absence of a facile emulsion and partitioning method has been a roadblock to full automation which is overcome with this new design. To demonstrate the utility and flexibility of the new design and emulsification method, cell lines transfected with a whole human genome CRISPR cas9 library were partitioned, libraries prepared, and sequencing completed. Whole transcriptome mRNA sensitivity was comparable between previous designs. Guide detection was sensitive with >99% guides detected per experiment.