GSM7918259: E14, biol rep 2, H3K9me3, CUT&RUN; Mus musculus; OTHER
Unfixed nuclei were extracted and bound to concanavalin A beads (EpiCypher), followed by an addition of primary antibody. For CUT&Tag, secondary antibody was applied, followed by an addition of pAG-Tn5 (EpiCypher). Tagmentation was then performed by an addition of magnesium. The DNA fragments were then extracted by 0.1% SDS. For CUT&RUN, pAG-MNase was applied, washed, and then activated by calcium chloride. Then DNA fragments were purified from supernatant using the column purification (EpiCypher). The CUT&Tag libraries were prepared with i5 and i7 primers (IDT) and Q5 High-Fidelity 2× Master Mix (NEB) and amplified using 15 cycles (RNAPIISer5-P and H3K27me3) or 17 cycles (IgG) on the thermocycler. The CUT&RUN libraries were prepared with CUT&RUN Library Prep Kit (EpiCypher) and amplified using 12 cycles on the thermocycler. Post amplification libraries were purified by using magnetic beads (Omega Bio-Tek), followed by a gel electrophoresis with 1% agarose gels (Invitrogen) and a column purification (Zymo Research). Libraries were validated using the High Sensitivity DNA kit (Agilent).