Snap‐frozen mouse liver (100 mg) from young and old wild‐type mice was used to prepare chromatin. ChIP and H3K14ac ChIP‐Seq were performed as described previously (Whitton et al., 2018 ). For Setdb1 and Kap1, a few modifications were incorporated. Particularly, sonication, using Diagenode Bioruptor Pico, was reduced to 11 cycles (30 s pulse on, 30 s pulse off) and libraries were sequenced using Illumina NextSeq 500 following the manufacturer's protocols. Rabbit polyclonal antibodies specific to KAP1 (Abcam, ab10483), SETDB1 (Proteintech, 11231–1‐AP), and anti‐acetyl‐histone‐H3 specific to Lys‐14 (Millipore, 07–353) were used for immunoprecipitation.
For sequential ChIP, the first immunoprecipitation was performed as described for ChIP (Whitton et al.,2018 ) with some modifications. After the washes, samples were eluted in 100 µl 1% SDS and 10 mM fresh dithiothreitol (DTT) twice for a total elution volume of 200 µl. After each elution, the samples were rotated at 37ºC for 15 min. After the second elution, samples were diluted in TE to 1.5 ml. Then, the second antibody was added and the second ChIP was performed as described previously (Whitton et al., 2018 ). Rabbit antihistone H3 (trimethyl K9) antibody (ab8898) and anti‐acetyl‐histone‐H3 specific to Lys‐14 (Millipore, 07–353) were used for immunoprecipitation.
For sequential ChIP, the first immunoprecipitation was performed as described for ChIP (Whitton et al.,