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Fig. 1 | Genome Biology

Fig. 1

From: Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network

Fig. 1

Profiling of Oct4 promoter-interacting lncRNAs by CRIST-seq. A Schematic diagram of the chromatin-lncRNA in situ reverse transcription trap sequencing (CRIST-Seq) assay. dCas9: Catalytically inactive CRISPR Cas9; FLAG: a tag octapeptide having the sequence motif DYKDDDDK that is attached to the N-terminal of Cas9; Oct4-gRNA: Cas9 guiding RNAs that target the Oct4 promoter. In iPSCs, Cas9-gRNA binds to the Oct4 promoter through a mechanism of base pairing between the gRNA and target DNA. After fixation, the Oct4 promoter-interacting RNAs were reverse transcribed into cDNAs in the isolated nuclei with biotin-dCTP. The Cas9 Oct4 promoter biotin-cDNA complex was immunoprecipitated by a Cas9-FLAG antibody, and biotin-cDNAs were further purified from genomic DNAs by biotin-streptavidin bead purification. The CRIST-captured cDNAs were profiled by Illumina library sequencing to identify the RNA components that regulate pluripotency. B Profiling pluripotency-associated lncRNAs by the combined CRIST-seq and RNA-Seq datasets. The Oct4-interacting lncRNAs identified by CRIST-seq were integrated with the dataset of RNA transcriptome sequencing. The combination of these two datasets identifies lncRNAs that not only interact with the Oct4 promoter but are also expressed differentially in reprogramming. C Integration of the RNA-Seq and CRIST-Seq datasets. RNA-Seq was initially used to identify the upregulated RNAs (>2-fold, p<0.05) in reprogramming. The upregulated RNAs were then integrated into the Oct4 and Sox2 CRIST-Seq datasets using a VENN program. The CRIST-Seq data were adjusted over the values of the IgG control and Cas9-gCT control. A cut-off threshold of peak enrichment FPKM>50 was arbitrarily set to select CRIST-Seq RNAs for VENN analysis. Integration of three datasets generated a list of 27 pluripotency-associated RNA candidates. D A list of 27 pluripotency-associated RNA (PALR) candidates identified by RNA-Seq and CRIST-Seq. The RNA candidates are ranked based on the RNA expression-fold from high (red) to low (blue) between fibroblasts (FBC) and iPSCs

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