title: GSM4417267: mESC_SMC3_rep1; Mus musculus; OTHER
library_construction_protocol: The DNA extraction follows CUT &RUN protocol SMC3 CUT&RUN in mESCs was conducted as previously described (Skene and Henikoff, 2017). Briefly, samples were resuspended by washing buffer (HEPES-KOH, pH = 7.5, 20mM; NaCl, 150mM; Spermidine, 0.5mM and with Roche complete protease inhibitor) and incubated with concanavalin-coated magnetic beads (Polyscience, 86057) at 23°C for 10 mins. Next, the samples were resuspended by antibody buffer (washing buffer plus digitonin (Thermo, Cat # BN2006), freshly pre-heated, 0.005%~0.01%, tested for each batch; EDTA, pH = 8.0, 2mM) with SMC3 antibody (Abcam, Cat #ab9263) diluted at ratio of 1:100. After being incubated at 4°C for overnight, the samples were resuspended by washing buffer with pA-MNase (to a final concentration of 700ng/mL) and incubated at 4°C for 3 hours. The targeted digestion was then performed with CaCl2 treatment and quenched by stop buffer. Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). The products were purified and size-selected with AMPure XP beads (Beckman Coulter, Cat # A63881).
attributes.tag: GEO Accession
attributes.value: GSM4417267