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Chromatin-associated RNAchIP and transcriptome analyses of the relationship between HP1g/CBX3 and RNA [RIP-seq]

ABSTRACT: RNA-seq and RNAchIP-seq experiments were performed on MEF-derived cell lines knock-out for the expression of HP1g/CBX3 (R cell line) or recomplemented for the expression of HP1g (HPg cell line). Cells were tested under normal growth or conditions of stress (p samples) by treatment with the phorbol ester PMA. Each experiment was performed in biological triplicates.

ORGANISM(S): Mus musculus

PROVIDER: GSE133265 | GEO | 2021/06/29

REPOSITORIES: GEO

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Project description:Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. We have found previously that activation of protein kinase C beta 2 (PRKCB2) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC, but the genetic program triggered by PRKCB2 activation that results in DC differentiation is only beginning to be characterized. Of the cPKC isoforms, only PRKCB2 was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. To examine early changes in gene expression following PRKCB2 activation, we employed the following cell lines: (1) the CD34(+) human acute myeloid leukemia derived cell line KG1, which undergoes DC differentiation following phorbol ester treatment; (2) KG1a, a spontaneously arising differentiation-resistant daughter cell line of KG1 that has lost PRKCB2 expression; (3) clones established from KG1a that stably express exogenous PRKCB2-GFP fusion proteins and are once again able to undergo DC differentiation (KG1a-PRKCB2-GFP Clone E9 and Clone E11). We examined changes in gene expression in these cells following treatment with the phorbol ester PMA (phorbol 12-myristate 13-acetate) for 2 hours. Since KG1 and KG1a differ in PRKCB2 expression but have similar expression of the other protein kinase C isoforms, this protocol will allow for the identification of genes regulated by PRKCB2 activation. KG1, KG1a, E9, and E11 were cultured for 2 hours in normal media +/- PMA (10 ng/ml). RNA was then isolated using RNeasy mini-columns (Qiagen) following the standard protocol. Samples were then sent to Expression Analysis, who carried out quality control, sample processing/labeling, hybridization to Affymetrix GeneChip arrays (Human U133 2.0 Plus), imaging, and analysis according to their standard protocols. Gene expression profiles were generated for each cell line in the presence and absence of phorbol ester stimulation, for a total of 12 samples (E9 +/- PMA and E11 +/- PMA were run in duplicate).

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Chromatin-associated RNAchIP and transcriptome analyses of the relationship between HP1g/CBX3 and RNA [RIP-seq]

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