scRNAseq of untagged cells
Samples were processed with the Chromium Next GEM Single Cell 3' HT Kit (10x Genomics, CG000416 Rev C). Pooled tag libraries treated with HyT36 were loaded across 3 GEM wells at 88 k cells each per timepoint with the goal of recovering 39 k singlets per well after filtering. Vehicle and untagged controls were loaded each into a single well at 30.5 k and 14.5 k cells, respectively, with the goal of recovering 17 k and 8 k singlets after filtering. Targeted amplification of sgRNA sequences from the CROPseq backbone was performed in order to reliably identify the tag in each cell. Sequencing libraries were prepared from three sequential PCR reactions starting from cDNA obtained during the 10x single cell processing protocol. The number of cycles for each PCR reaction was optimized by qPCR with EvaGreen on a CFX Opus 96 Real-Time PCR System.