GSM7911394: Control_1, DRIP, LUAD, PC-9; Homo sapiens; OTHER
Genomic DNA Extraction & Fragmentation: Total nucleic acids were extracted from cells by SDS/proteinase K treatment followed by phenol-chloroform extraction and ethanol precipitation. More than 10 µg 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). DRIP immunoprecipitation: The fragmented DNA was divided equally into “IgG”, and “RIP” aliquots. Each of the two aliquots was mixed with 1 µL the synthetic RNA̢DNA hybrid spike-ins. “DRIP” was immunoprecipitated with 20 µg S9.6 antibody bound to protein G resin, “lgG” was immunoprecipitated with 20 µg non-immune IgG instead. The reactions were incubated at 4°C for overnight with head-over-tail rotation. The beads were washed three times with binding buffer and eluted in 250 µL elution buffer at 50°C for 30 min with rotation. The eluted DNAs were cleaned up by phenol chloroform and eluted in 15 µl ddH2O. DRIP-seq library preparation: Followed by second DNA strand synthesis. The materials were cleaned up with 1.8X volume of DNA Beads and ligate “A” and adaptor. Add 10 µl ddH2O and 35 µl PEG/NaCl (1:1) to clean up and eluted with 10 µl ddH2O. Add 11 µl KAPA HiFi HotStart ReadyMix (2X) and 1 µl primers to amplify library. Each DNA library was amplified 9-20 cycles depending on the starting material amount. The library DNA was purified by 0.8X volume of DNA Beads. The library concentration and size distribution were checked by Qubit and agarose gel.