title: GSM6564312: Naive_ESC_MLL3KO_MLL4KO_H3K27a_rep1; Mus musculus; OTHER
library_name: GSM6564312
library_construction_protocol: CUT&TAG was conducted using the protocol from (Kaya-Okur et al. 2020) with the following modifications: freshly trypsinized cells were bound to Concanavalin A beads at a ratio of 2e5 cells/7ul beads in CR wash at room temp. We used 2e5 cells as input per sample. Bead-bound cells were then incubated rotating overnight at 4C in CT Antibody buffer (CR Wash with 0.05% Digitonin, 2mM EDTA, 1mg/mL BSA) containing primary antibody. We used the following primary antibodies for CUT&TAG: 1:100 Rabbit anti-H3K27a (Abcam, ab4729), 1:100 Rabbit anti-H3K4m3 (Abcam, ab8580), 1:100 Rabbit IgG isotype control (Abcam, ab171870). 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 4C for 1 hour rotating at 4C. Samples were then incubated for 1 hour at 4C with 50ul of approximately 25nM homemade pA-Tn5 in CT Dig300 wash buffer (20mM HEPES, 300mM NaCl, 0.01% Digitonin, 0.5mM 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 37C in a thermocycler by adding MgCl2 to 10mM final concentration in 50uL volume. The tagmentation reaction was quenched immediately afterwards by adding 1.6ul of 0.5M EDTA, 1ul of 10mg/mL Proteinase K, and 1ul of 5% SDS. Samples were then incubated at 55C 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 100bp. 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 >250bp and eluted in 0.1X TE. Quality and concentration of libraries were determined by an Agilent 4200 Tapestation with D1000 reagents before pooling for sequencing.