Unknown,Transcriptomics,Genomics,Proteomics

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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 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application. RNA-Seq of 181 single-cell samples from 8 FACS sorted cell types: 1. endothelial cells (samples E11.0_EC_xxxx. CD31+ VE-cadherin+CD41-CD43-CD45-Ter119-); 2. T1 pre-HSCs (samples E11.0_T1_xxxx. CD31+CD45-CD41low c-Kit+CD201high); 3. T1 CD201- cells (samples E11.0_T1CD201neg_xxxx, CD31+CD45-CD41low c-Kit+CD201low/-) ; 4. T2 pre-HSCs (samples E11.0_T2_35xx. CD31+CD45+c-Kit+CD201high), 5. T2 CD41low (samples E11.0_T2_21xx, E11.0_T2_24xx and E11.0_T2_27xx. CD31+CD45+CD41low); 6. E12 HSCs (samples E12.5_FL_xxxx. Lin-Sca-1+Mac-1lowCD201+); 7. E14 HSCs (samples E14.5_FL_xxxx. CD45+CD150+CD48-CD201+); 8. Adult HSCs (samples Adult_HSC_xxxx. CD45+CD150+CD48-CD201+). ECs, T1 pre-HSCs, T1 CD201- cells, T2 pre-HSCs, T2 CD41low cells were sorted from E11 AGM region. Mature HSCs were sorted from E12 or E14 fetal liver and adult bonemarrow.

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

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