GSM3244961: Input DNA rep4; Mus musculus; ChIP-Seq
Genome wide analysis of cromatin states in lung tumors induced by K-RasLSL-G12D and K-RasLSL-G12D;Mll4fl/fl  mice after Ad-CMV-Cre treatment.  Six histone modifications (H3K27Ac,H3K4me1, H3K79me2, H3K9me3, H3K4me3 and H3K27me3) were mapped by ChIP-seq. ChIP assays were performed as previously described (Terranova et al., 2018).Briefly, lung tumor tissue (3mg per antibody) were cut into 1mm pieces, homogenized using MACS dissociator using optimized conditions and cross-linked using 1% paraformaldehyde for 10 min at 37oC. Crosslinking was then stopped by adding 0.125M glycine for 5mins, and tissues were washed with PBS and stored at -80oC. Next day tissues were thawed on ice and lysed with ChIP harvest buffer (12 mM Tris-Cl, 0.1x PBS, 6 mM EDTA, 0.5% SDS) for 10min on ice. Sonication conditions were optimized for lung tumor tissues using bioruptor sonicator to achieve a shear length of 250- 500bp. Antibody-dynabead mixtures were incubated for 1hr at 4oC and tissue extracts were then incubated overnight with antibody-dynabead mixtures. After overnight incubation, immunecomplexes were washed in following order: 5 times with RIPA buffer, twice with RIPA-500 (RIPA with 500mM NaCl) and twice with LiCl wash buffer (10mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 250mM LiCl, 0.5% NP-40, 0.1% DOC). For decrosslinking and elution, immunecomplexes were incubated overnight at 65oC in direct elution buffer (10mM Tris-Cl pH8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS). Eluted DNA was then treated with Proteinase K (20mg/ml) and RNaseA and DNA cleaned up was done using SPRI beads (Beckman-Coulter). Library was prepared as described earlier (Terranova et al., 2018) using NEB adapters. Libraries were multiplexed together and sequencing was performed in Hiseq2000 Library was prepared as described earlier (Terranova et al., 2018) using NEB adapters
GEO Accession
GSM3244961