Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Fixed cells were lysed during 20 min in ice-cold lysis buffer (100mM NaCl, 66mM Tris HCl pH8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with proteases inhibitors, passed through a 21G needle and sonicated using a Bioruptor nextGen (Diagenode) to obtain chromatin frgament size of 250-500bp. Sonicated chromatin was centrifuged at 14000rpm at 4ºC for 10 min and the supernatant used for subsequent steps. In parallel, and independently, Drosophila S2 cells were fixed, lysed and sonicated as described for Hela S3 cells. After sonication, HeLa S3 input chromatin required for the analysis of all time-points from the same time course experiment was mixed with Drosophila S2 chromatin (2.5% of total chromatin). Chromatin associated to each histone mark was obtained by incubating sonicated chromatin with the corresponding antibody. Libraries were costructed using the KAPA Hyperprep kit following manufacturer's instruction, except that 1.25 µM of Illumina compatible indexed adapters (Pentabase) were ligated to A-tailed DNA. Following adapter ligation biotin-TEG-azide (Berry&Associates) was linked to EdU in Click reaction. Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific) and subjected to PCR amplification following the KAPA Hyperprep kit protocol.