title: GSM3465913: wt_H3K9me2_ChIP; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: 2 g of 2-weeks-old seedlings were cross-linked with 32 ml of crosslinking buffer (0.4 M sucrose, 10 mM tris-HCl pH 8.0, 1 mM EDTA and 1% formaldehyde (Thermo Scientific, cat. No. 28906)) for 10 min under vacuum. Cross-linking was quenched by applying vacuum for 5 min with 2 ml 2 M glycine. Cross-linked seedlings were washed with distilled water, pat dried with paper towels, frozen and ground in liquid nitrogen with pestle and mortar. Ground tissues were re-suspended in 20 ml of nuclei isolation buffer at 4 °C (NIB; 0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, 1X complete EDTA-free protease inhibitor cocktail (Roche, cat. No. 11873580001)). Homogenized slurry was filtered through Miracloth (Millipore, cat. No. 475855) and centrifuged at 6,000 rpm for 20 min at 4 °C to pull-down nuclei. After discarding supernatant, cell pellets were re-suspended in 1ml 4 °C nuclei lysis buffer (NLB; 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 % SDS, 1 % Triton X-100, 1X complete EDTA-free protease inhibitor). 500 ml nuclei solution was sonicated and centrifuged for 10 min at 13,000 rpm in table-top centrifuge (4 °C). Sonicated chromatin in supernatant was transferred and diluted 5 times with dilution buffer (NLB without SDS and Triton X-100). 200 ml of diluted chromatin was incubated with 7 ml of antibodies against H3K9me2 (Abcam, cat. No. AB1220) overnight with rotation at 4 °C. 45 ml of pre-cleared protein G dynabeads (Invitrogen, 10003D) were added and incubated for 4 hours with rotation at 4 °C. 20 ml of diluted chromatin solution was kept for input control. Chromatin bound to the antibodies were washed 4 times with low salt wash buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.2% SDS, 0.5% Triton X-100, 2 mM EDTA) and one time with TE buffer (1 mM EDTA and 10 mM Tris-HCl pH 8.0) at 4 °C. Washed chromatin were re-suspended in 200 ml elution buffer (1% SDS), and incubated at 65 °C for 5 hrs with 8 ml of 5M NaCl (vortex for 1 hr). To digest proteins bound to gDNA, samples were incubated 1 hr at 55 °C with 4 ml proteinase K (Thermo, cat. No. EO0492), 4 ml of 0.5 M EDTA, 8 ml of 1 M tris-HCl pH 8. After reverse-crosslinking, 220 ml P:C:I, 500 ml ethanol, 20 ml 3M sodium acetate (pH 5.2), and 2 ml glycogen were added and incubated overnight at -80 °C to extract DNA.
attributes.tag: GEO Accession
attributes.value: GSM3465913