GSE97020
Loss of Asxl2 leads to myeloid malignancies in mice. [ChIP-Seq]
LK cells of each mouse genotype were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by shearing with Bioruptor Pico with water cooler (Diagenode, Seraing, Belgium). The DNA was sheared to an average length of 300-500 bp. Genomic DNA regions of interest were isolated using antibodies against H3K27ac (Diagenode, C15410196), H3K4me1 (C15410194), and H3K4me2 (Abcam, ab32356). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. After the measurement of DNA concentration with Qubit3.0, the libraries were prepared and sequenced on an Illumina NextSeq 500. Overall design: To delineate the effect of ASXL2 in the maintenance of HSC self-renewal and cell fates, we performed RNA-seq on LK cells isolated from 6-week-old WT and Asxl2-/- mice with three biological replicates. To explore the impact of ASXL2 loss on histone modifications in HSC/HPCs, we performed Chromatin Immunoprecipitation (ChIP) assays followed by sequencing (ChIP-seq) using antibodies against H3K4me1, H3K4me2, and H3K27ac.