title: 7day_rep1_IP_R1_DIP-Seq
design: Total nucleic acids were extracted from cells and tissues by SDS/proteinase K treatment followed by phenol chloroform extraction and ethanol precipitation. Typically more than 10 g genomic DNA for each sample was fragmented by ultrasound (Perform 10-15 cycles of 30 s ON/ 30 s OFF, spin after 3, 6, 9, and 12 cycles to ensure homogeneous sonication). The fragments length are limited within 500bp as possible.Fragmented DNA was divided equally into IgG, and DRIPc aliquots. Each of the two aliquots was mixed with 1 L of synthetic RNADNA hybrid spike-ins. DRIPc was immunoprecipitated with 20 g S9.6 antibody bound to protein G resin, lgG was immunoprecipitated with 20 g non-immune IgG instead. The reactions were incubated at 4C for overnight with head-over-tail rotation. Incubated resin was washed three times with 800 L binding buffer and eluted in 250 L elution buffer at 50C for 30 min with rotation. Eluted DNAs were cleaned up by phenol chloroform and eluted in 15 L ddH2O. Followed by second DNA strand synthesis with dUTP, dsDNA was cleaned up with 1.8 volume of DNA Beads and ligate A and adaptor. Add 10 L ddH2O and 35 uL PEG/NaCl (1:1) to clean up and eluted with 10 L ddH2O. Add 11 L KAPA HiFi HotStart ReadyMix (2X) and 1 uL primers to amplify library. Each DNAlibrary was amplified 9-20 cycles depending on the starting material amount. Library was purified by 0.8  volume of DNA Beads. The library concentration and size distribution were checked by Qubit and 2100.