S2x focused ultrasonicator

BMDMs derived from WT or Setdb2^GT/GT mice were either treated with poly(I:C) for 2h or left untreated. The same number of cells was subsequently harvested, fixed with 1% formaldehyde for 10min at RT, and lysed in 1% SDS buffer. Chromatin was sheared to an average size of 300bp using S2X Focused-ultrasonicator (Covaris), and the amount was adjusted using ND-1000 spectrophotometer (NanoDrop) measurement to match WT and Setdb2^GT/GT samples before histone mark ChIPs. The prepared chromatin of WT or WT and Setdb2^GT/GT samples were subjected to ChIP with anti-Setdb2 clone 7H7F11 (see above), anti-H3K9me3 (Abcam Ab8898) or anti-H3K9acetyl (Millipore #07-352) respectively following a procedure as described previously ^{50 (link)} that was modified by the use of magnetic Dynabeads Protein-G beads (Life Technologies). For mock ChIP controls, empty beads were used. For mock controls in Setdb2 ChIP, the medium used for culturing the hybridoma 7H7F11 was added instead. The ChIP efficiency was controlled by quantitative real-time PCR analysis using the primers for: Cxcl1 promoter region, F 5’-CCTCTTCACATGCCTCCCTG-3’ and R 5’-CGGGGATGGAAGCTTGTCTT-3’; Cxcl1 exon 1 region, F 5’-GTTCCAGCACTCCAGACTCC-3’ and R 5’-AGTGGCGAGACCTACCTGT-3’; Actb promoter region, F 5’-CCTCTGGGTGTGGATGTCAC-3’ and R 5’-TGTCCATTCAATCCAGGCCC-3’.