GSM8528410: EA18.1_CUTTag_ESC_H3K27me3_DMSO_1d_rep1; Mus musculus; OTHER
CUT&Tag was conducted using the protocol from Kaya-Okur et al. 2020 with the following modifications: freshly collected cells were bound to Concanavalin A beads at a ratio of 2e5 cells/7 μL beads in CR wash at RT. At least 1e5 cells were used as input per sample. Bead-bound cells were then incubated rotating overnight at 4°C in CT Antibody buffer (CR Wash with 0.05% Digitonin, 2 mM EDTA, 1 mg/mL BSA) containing primary antibody. After primary, samples were washed 3 times for 5 minutes each using CR Dig-wash buffer and resuspended in 1:100 secondary antibody (Guinea pig anti-rabbit, Antibodies Online #ABIN101961) in CR Dig-wash buffer at 4°C for 1 hour rotating at 4°C. Samples were then incubated for 1 hour at 4°C with 50 μL of approximately 25 nM homemade pA-Tn5 in CT Dig300 wash buffer (20 mM HEPES, 300 mM NaCl, 0.01% Digitonin, 0.5 mM Spermidine with Roche cOmplete protease inhibitors added). Recombinant Tn5 was purified and loaded with adapters as previously described (Kaya-Okur et al. 2019). After Tn5 incubation, samples were washed 3 times for 5 minutes each with CT Dig300 wash buffer. Tagmentation was then initiated for 1hr at 37°C in a thermocycler by adding MgCl2 to 10 mM final concentration in 50 μL volume. The tagmentation reaction was quenched immediately afterwards by adding 1.6 μL l of 0.5 M EDTA, 1 μL of 10 mg/mL Proteinase K, and 1 μL of 5% SDS. Samples were then incubated at 55°C for 2 hours in a thermocycler to denature Tn5 and solubilize tagmented chromatin. After incubation, samples were magnetized, and the supernatant was transferred to new wells where SPRI bead purification was performed using homemade beads to select all DNA fragment lengths larger than 100 bp. Samples were eluted in 0.1X TE Approximately half of each sample was used for library preparation using NEBNext HIFI Polymerase with custom indices synthesized by IDT. An appropriate number of cycles for each target was chosen to prevent overamplification bias. After amplification, libraries were purified with 1.2X homemade SPRI beads to select for fragments >250 bp and eluted in 0.1X TE.