GSM8769281: MT_aux_NMMb_1_S25; Mus musculus; OTHER
The isolated nuclei from mESCs were mixed with activated Concanavalin A (ConA)-coated magnetic beads (Cat# 21-1401, Epicypher) and incubated for 10 minutes at room temperature. Upon nuclei extraction and binding nuclei to activated beads, incubation steps were completed in a modified Digitonin 150 buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1x protease/phosphatase inhibitor) until the tagmentation step. This modification, in which sodium chloride was replaced by potassium chloride, was to optimize for stabilization of G4 structures during prolonged incubation steps. Other buffers were prepared and used according to manufacturer's protocol (Epicypher CUT & Tag). The nuclei-conjugated ConA bead complexes were incubated overnight at 4 °C with primary antibody (CTCF, BG4, NMM-Bio, or control NMM) in the modified Digitonin 150 buffer. After primary incubation, nuclei-bead complexes were incubated with secondary anti-biotin antibody (Cat# ab53494, Abcam) in the modified Digitonin 150 buffer for 1 hour at room temperature, followed by tagmentation using CUTANA pAG-Tn5 (Cat# 15-1117, Epicypher). Targeted chromatin tagmented products were column purified using a DNA Clean & Concentrator Kit (Cat# D4013, Zymo Research). DNA libraries were amplified with the following thermocycling conditions: initial denaturation at 98 °C for 30 seconds, 18 cycles of denaturation at 98 °C for 10 seconds, annealing at 63 °C for 30 seconds, and extension at 72 °C for 1 minutes, followed by a final extension at 72 °C for 1 minutes. After PCR amplification, the libraries were column purified once again to be quantified and qualified before pooling for sequencing. Purified libraries were sequenced on Illumina NovaSeq 6000.