Transcriptomics

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Ontogenic changes in hematopoietic hierarchy determine pediatric specificity and disease phenotype in fusion oncogene-driven myeloid leukemia-ATACseq


ABSTRACT: ATACseq analyses between iEG (ETO2-GLIS2) and CTRL fetal primary cells and myeloid or CD41 iEG cells lines. Briefly, cells were isolated from iEG mice and cultured for 24 hours in RPMI supplemented with 10% FBS, cytokines (mIL3, mIL6, mSCF, mTPO, mFLT3l) and 100 ng/ml Doxycyclin. After cell lysis, transposition and purification step, the transposed DNA fragments were amplified by polymerase chain reaction (PCR) between 12 and 18 depending on the number of cells at the beginning (50,000 to 6,000) using adapters from the Nextera index kit (illumina). PCR purification was performed using Agencourt AMPure XP magnetic beads (Beckman Coulter A63880) in order to remove large fragments and remaining primers. Library quality was assessed using an Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent Technologies 5067-4626). Libraries were sequenced using Novaseq-6000 sequencer (Illumina) (50bp paired-end reads). Quality control of reads was performed using FastQC 0.11.7 and multiQC 1.5. The reads were aligned to the reference genome mm10 with bwa (aln 0.7.17). After alignment, we removed reads mapping to the mitochondrial genome, PCR duplicate reads and reads with a mapping quality lower than 20 using samtools (v 1.9). Final read counts for all mouse datasets ranged from 37 to 128 million reads. Mapped reads were normalized to bins per million (BPM) and were converted to bigwig format using deeptools (v3.2.0). Peak calling, differential analysis, annotation and motif analysis was performed using macs2 (V 2.1.2), Diffbind R package (v 2.8.0 in R-3.5.1 with threshold log2(1.5)), and homer (v4.10.4, annotatePeak.pl and findMotifsGenome.pl) respectively.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus  

SUBMITTER: Thomas MERCHER  

PROVIDER: E-MTAB-8375 | ArrayExpress | 2019-10-15

SECONDARY ACCESSION(S): ERP117530

REPOSITORIES: ArrayExpress, ENA

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