Next Generation Sequencing Analysis of Transcriptomes of the HEK293T-Cas9 cells transfected with plasmid containing programmed scaffold RNA

By performing RNA sequencing (RNA-seq) analysis on the HEK293T-Cas9 cells which transfected with plasmid containing programmed scaffold RNA for non-targeting or for targeting Hemoglobin subunit gamma-1 (HBG1) we want to investigate whether the scaffold RNA and the endogenous mRNA could be captured in regular reverse transcription reaction. RNA-seq analysis showed that we can capture the scaffold RNA together with the endogenous mRNA  precisely. Our framework offered strategies that can be conveniently applied to study the genotype and mRNA profiles after CRISPR-mediated genome perturbation with single-cell transcriptional readout We incorporated a poly(A)-like capture sequence (8A8G) into the gRNA scaffold, thus the gRNA transcript and endogenous mRNA can be recognized by oligo(dT) in the reverse transcription and characterized using various RNA-seq or single-cell RNA-seq platforms. HNT-8A8G: CRISPR activation targeting a non-target control with the 8A8G scaffold; HBG1-8A8G: CRISPR activation targeting HBG1 with the 8A8G scaffold; HBG1-WT: CRISPR activation targeting HBG1 but with WT-SAM scaffold.