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Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia

ABSTRACT: Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia

PROVIDER: PRJNA669859 | ENA |

REPOSITORIES: ENA

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Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia

Project description:Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the Mixed Lineage Leukemia-1 gene, which encodes the KMT2A lysine methyltransferase. The most common translocations produce in-frame fusions of KMT2A to trans-activation domains of chromatin regulatory proteins. Here we develop a strategy to map the genome-wide occupancy of oncogenic KMT2A fusion proteins in primary patient samples regardless of fusion partner. By modifying the versatile CUT&Tag method for full automation we identify common and tumor-specific patterns of aberrant chromatin regulation induced by different KMT2A fusion proteins. Integration of automated and single-cell CUT&Tag uncovers lineage heterogeneity within patient samples and provides an attractive avenue for future diagnostics.

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Project description:We performed the cleavage under targets and tagmentation (Cut & tag) assay followed by sequencing enriched DNA fragments to reveal the direct downstream targets of Pbx1. Firstly, we overexpressed Pbx1b with Pbx1b-IRES-GFP retrovirus in murine peripheral B cells to ensure the yields of DNA fragments. CUT & tag libraries were generated following instructions of the manufacturer’s protocol (Vazyme; cat TD901-01) and the Pbx1 antibody (CST; cat 4342) was used for signal enrichment.

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Low-cost high-resolution chromatin profiling with CUT&Tag

Project description:We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.

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