title: GSM4217468: METTL3 binding in senescent cells; Homo sapiens; OTHER
library_construction_protocol: For cut-and-run cells were harvested, and gently washed twice using wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and EDTA-free Protease Inhibitor Cocktail). Cells were then incubated with the antibody overnight in the buffer (Wash buffer supplemented with 0.05% digitonin and 2 mM EDTA) at 4°C. The next day, supernatant was removed by centrifuge and cell pellets were washed once with Dig-wash buffer (Wash buffer containing 0.05% digitonin). Cell pellets were then incubated with Protein A-MNase (700 ng/ml in Dig-wash buffer) for 1 hour by rotation at 4°C. After three times of wash, cell pellets were resuspended in 100 l Dig-wash buffer with 2 l 100 mM CaCl2, incubated at 0°C for 30 mins, and then reactions were stopped by addition of 100 l 2 x STOP buffer (340 mM NaCl, 20 mM EDTA pH 8.0, 4 mM EGTA, 0.05% digitonin, 50 g/ml RNase A, 50 g/ml glycogen and 2 pg/ml heterologous spike-in yeast DNA). For CHIP-seq, DNA was eluted by incubation of the beads with TE buffer plus 1% SDS for 15 mins at 65 °C. The samples were then incubated overnight at 65 °C to reverse crosslinking. The next day, the samples were digested with proteinase K and the DNA was purified using a Wizard SV Gel and PCR Clean Up kit (Promega) 10 ng of ChIP DNA was used for ChIP-seq library construction using NEBNext Ultra DNA Library Prep Kit (NEB, E7645)
attributes.tag: GEO Accession
attributes.value: GSM4217468