GSM7928234: ckit+ WT ms BM, HOXA9, replicate 2, CUT&RUN; Mus musculus; OTHER
CUT&RUN was performed according to Epicypher CUT&amp;RUN Protocol V2.0. Briefly, 0.5 million ckit + HSPCs from WT BL6 mice or Ctnnb1-Biotin-3xFLAG knockin mice (JAX:029511) were rinsed with wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and 1× Complete Protease Inhibitor Cocktail) and incubated with the activated Concanavalin A (ConA) beads (Bangs Laboratories BP531) at RT for 10 min. 0.5 μl antibody (HOXA9 Atlas HPA061982, FLAG Genscript A00170, IgG Cell Signaling Technology 3900S DA1E) was added and incubated at 4 °C overnight. After permeabilization with digitonin buffer (wash buffer plus 0.01% digitonin), 20 ng pAG-MNase (kindly provided by Dr Paul Lavender, King's College London) was added and bound to antibody-labelled chromatin. MNase was subsequently activated by addition of CaCl 2 to cleave and release antibody-bound chromatin. DNA purification was performed using Monarch PCR cleanup kit (NEB, Monarch PCR &amp; DNA Cleanup Kit, T1030). DNA libraries were prepared using the NEB Ultra II DNA Library Prep Kit (NEB #E7103) and sequencing was carried out by Novogene UK.