title: GSM4453145: P3_DKO_H3K9me2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were suspended in 25 μl of 0.3 M sucrose-containing buffer 1 (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 15 mM Tris-HCl pH 7.5, and protease inhibitor cocktail). The cells were then lysed following the addition of 0.3 M sucrose-containing buffer 1 (25 μl) with 0.8% NP40 on ice for 10 min; 1.2 M sucrose-containing buffer 1 (400 μl) was added and the chromatin was collected as pellets by centrifugation. The pellets were digested with micrococcal nuclease (0.05 U, Takara) in 10 μl of digestion buffer (0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris-HCl pH 7.5) by vortexing at 37°C for 15 min; digestion was stopped with EDTA. The supernatant was obtained by centrifugation and incubated with anti-H3K9me2-conjugated magnetic beads (Dynabeads Protein G, Invitrogen) in 50 μl of incubation buffer (50 mM NaCl, 5 mM EDTA, 0.1% NP40, 20 mM Tris-HCl pH 7.5) at 4°C for 6 h. Then, DNA was extracted from the immune complex according to the standard protocol. DNA from input and ChIP fractions was processed using SMARTer ThruPLEX DNA-seq Kit (TaKaRa) and sequenced using the Illumina HiSeq 1500 system according to the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4453145