Profiling lncRNAs in the regulatory elements of target genes by chromatin in situ reverse transcription trap sequencing

“chromatin-RNA in situ reverse transcription trap sequencing” (CRIST-Seq) approach was used to profile the RNA interaction network in the promoters of stem cell core factors Sox2 and Oct4 Overall design: Induced pluripotent stem cells (iPSCs) were transfected with CRISPR Cas9 and four gRNAs targeting the Oct4 and Sox2 promoters (Oct4-gRNA and Sox2-gRNA). As the Cas9 control, cells were transfected with CRISPR Cas9 and two random control gRNAs (gCT). Cells were crosslinked with formaldehyde to fix the chromatin structure. RNAs in the chromatin complex of the promoters were in situ reverse transcribed with biotin-dCTP. After immunoprecipitation with Cas9-FLAG antibody, the biotin-cDNAs were separated from genomic DNAs by streptavidin bead purification. The purified biotin cDNAs were used for Illumina library sequencing. An anti-IgG antibody was used as the background control for CRIST immunoprecipitation (IgG-Ct).