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RNA quantitative sequencing in bleomycin-treated p300 knockout primary ATII cells

ABSTRACT: RNA quantitative sequencing in bleomycin-treated p300 knockout primary ATII cells

PROVIDER: PRJNA785994 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17124546.fastq.gz	Fastqsanger.gz
	SRR17124547.fastq.gz	Fastqsanger.gz
	SRR17124548.fastq.gz	Fastqsanger.gz
	SRR17124549.fastq.gz	Fastqsanger.gz
	SRR17124550.fastq.gz	Fastqsanger.gz

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RNA quantitative sequencing in bleomycin-treated p300 knockout primary ATII cells

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Project description:Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

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Project description:Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was detemined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating 'tracks' for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the wide spread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.

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