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. Overall design: 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.