GSM5994281: Hematopoietic stem cells- RxRa CUT&RUN; Mus musculus; OTHER
Sorted and Bexarotene treated HSC and MPP were washed twice with wash buffer [20 mM HEPES; PH-7.5, 150mM NaCl, 0.5mM spermidine] containing protease inhibitor cocktail (Roche, Catalog. 11873580001). Cells were bound to Concanavalin A magnetic beads (Bangs Laboratories, Inc. Catalog. BP531) followed by permeabilization and antibody binding (RxRa antibody, Catalog. 21218-1-AP, Proteintech)  at 1:50 dilution in wash buffer containing 0.025% digitonin. Antibody bound cells were treated with pA-MNase (700 ng/mL) for 1h at 40C followed by targeted digestion in presence of CaCl2 at 00C for 30 min. The digestion reaction was stopped by adding STOP buffer containing 20 mM EDTA and 4mM EGTA.  The MNase digested DNA fragments were released at 370C, and extracted using QIAGEN MinElute Reaction Cleanup kits (Catalog. 28204). The CUT&RUNseq libraries were prepared by using NEBNext Ultra II DNA library preparation kit for Illumina (Catalog. E7103S) and NEBNext multiplex Oligoes for Illumina (Catalog. E7335S). The libraries were run on Illumina sequencer NovaSeq6000 with paired end sequencing.