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Next Generation Sequencing profile of leukemia from distinct cells-of-origin [ATAC-seq]

ABSTRACT: The goal of this study is to identify genomic signatures predicitve of cell-of-origin in acute myeloid leukemia Cryopreserved leukemic bone marrow samples were thawed, and 50,000 LSCs (L-GMP; GFP+ Lin- Sca-1- c-Kit+ CD34+ FcγR+) were sorted by FACSAria (BD). Samples were placed at 37oC and 5% CO2 in IMDM plus 10% fetal calf serum for 30 minutes, then harvested, washed with 1X PBS, and ATAC-seq libraries were prepared. Quantitative PCR using the Library Quantitation kit (Kapa Biosystems) was used to estimate library concentrations. Libraries were sequenced on the Illumina HiSeq 2000 platform generating 2x150bp paired end reads at a sequencing depth of ~30-50 million reads per sample. Libraries were sequenced on the Illumina HiSeq 2000 platform.

ORGANISM(S): Mus musculus

PROVIDER: GSE81805 | GEO | 2016/07/12

SECONDARY ACCESSION(S): PRJNA322591

REPOSITORIES: GEO

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Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit or Ovation Ultralow system (NuGen) according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments.

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Next Generation Sequencing profile of leukemia from distinct cells-of-origin [ATAC-seq]

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