Combined RNA-seq and RAT-seq mapping of long noncoding RNAs in pluripotent reprogramming

Abstract

Pluripotent stem cells hold great investigative potential for developmental biology and regenerative medicine. Recent studies suggest that long noncoding RNAs (lncRNAs) may function as key regulators of the maintenance and the lineage differentiation of stem cells. However, the underlying mechanisms by which lncRNAs affect the reprogramming process of somatic cells into pluripotent cells remain largely unknown. Using fibroblasts and induced pluripotent stem cells (iPSCs) at different stages of reprogramming, we performed RNA transcriptome sequencing (RNA-Seq) to identify lncRNAs that are differentially-expressed in association with pluripotency. An RNA reverse transcription-associated trap sequencing (RAT-seq) approach was then utilized to generate a database to map the regulatory element network for lncRNA candidates. Integration of these datasets can facilitate the identification of functional lncRNAs that are associated with reprogramming. Identification of lncRNAs that regulate pluripotency may lead to new strategies for enhancing iPSC induction in regenerative medicine.

Osblr8 orchestrates intrachromosomal loop structure required for maintaining stem cell pluripotency

“…We reasoned that an ideal pluripotent lncRNA candidate should also become activated in reprogramming. Therefore, we collected cells at different stages of reprogramming [27,28], and RNA-Seq was performed to identify RNAs that were differentially expressed in association with reprogramming [25]. To identify the pluripotency-associated lncRNAs candidates, we integrated the Oct4 and Sox2 CRIST lncRNA data with the RNA-Seq data (Fig.1B).…”

“…After CRIST-seq, the called peaks that overlapped with the IgG control enriched regions were removed, and the CRIST-Seq signal intensities were further normalized over that of the non-targeting Cas9 gCT control using parameters of fold-change difference ≥2 and p-value < 0.05, with false discovery rate (FDR) <0.1. The adjusted CRIST-Seq data were then used for mapping the Oct4 and Sox2 RNA interactions [24,25].…”

“…The isolated iPSC colonies were characterized by immunostaining stem cell markers, alkaline phosphatase staining, karyotype analysis, and teratoma formation. The fibroblast-like cells that expressed OSKM but were not reprogrammed were termed "non-iPSCs" and used in parallel with iPSCs in the study [8,25]. The lncRNAs that are differentially expressed in reprogramming were identified by RNA-seq.…”

“…We have used an RNA reverse transcription-associated capture sequencing (RAT-seq) approach to characterize functional lncRNAs that interact with the Oct4 promoter [24], a key transcription factor required for reprogramming somatic cells into iPSCs. The data from conventional RNA-seq were combined with RAT-seq to identify differentially-expressed lncRNAs that may be associated with intrachromosomal looping [25]. Using this strategy, we identified a series of functional lncRNA candidates that are associated with pluripotency [24,25].…”

“…The data from conventional RNA-seq were combined with RAT-seq to identify differentially-expressed lncRNAs that may be associated with intrachromosomal looping [25]. Using this strategy, we identified a series of functional lncRNA candidates that are associated with pluripotency [24,25]. We characterized Peblr20, an Oct4 enhancer binding lncRNA, as an essential chromatin factor for the maintenance of stem cell pluripotency.…”