All materials used in this study are commercially available, and the detailed information can be found in the Key Resources Table
All materials used in this study are commercially available, and the detailed information can be found in the Key Resources Table. Data and Code Availability This study did not generate new data or code. Acknowledgments We thank all members from the Chen lab and the Zhang lab for the help with the experiments. GSK 2830371 details on how to check sonication and perform an initial test on antibodies. Sonication serves two purposes: to solubilize chromatin and to fragment DNA to a size range that is suitable for next generation sequencing. However, during the sonication process, we found the epitopes of some proteins can be destroyed as well. The main purpose here is to find a balance between getting the DNA to the right range and maintain the protein integrity at the same time. This is the most variable process in the entire protocol, because sonication is usually highly dependent on the machine in use. We have successful experience using both probe sonicators and water bath sonicators. both the cell concentration and the volume can influence sonication results. We routinely used 300?L Sonication/IP Buffer to resuspend cells from 105 to 5? 106. When more than 5? 106 cells are used, we scale up the volume Rabbit polyclonal to FN1 to maintain a concentration of 5? 106 per 300?L volume. If the final volume exceeds the recommendation of the sonicator, make aliquots to perform sonication. When using the Bioruptor water bath sonicator, the maximum recommended volume in a 1.5?mL Eppendorf tube is 300?L. Therefore, it is not possible to just use one tube for the entire time course due the volume needed for the DNA and protein analysis. We normally prepare multiple tubes, one for each time point. all the chemicals and solutions listed in the Key Resources Table can be purchased from different suppliers for your own convenience, provided they are all molecular biology grade. Other systems such as Bioruptor Plus, Covaris, and probe sonicators. Any magnet with tube racks. Any qPCR machine. Other systems such as Agilent TapeStation, Fragment Analyzer, Caliper LabChip GX. at 4C for 10?min. b. During the 10-min centrifugation time, wash the antibody-beads complex from step 4d three times with 500?L Blocking Solution in the same way as described in actions 4a and 4b. c. Save 2?L supernatant from step 7a, and store in ?20C as the input sample, and transfer the rest supernatant to the washed antibody-beads complex. Incubate overnight (12C20 h) at 4C on a rotator. There should be very tiny or no visible pellet after the GSK 2830371 centrifugation at step 7a. The washes from step 11 to 14 are performed in the same way as described in step 10. for 30 s. 16. Put the tube on DynaMag-2 and remove trace of Tris-HCl. 17. Resuspend the beads thoroughly with 30?L tagmentation mix, which consists of 15?L 2 TD Buffer?+ 14?L ddH2O?+ 1?L Tn5. The Tn5 can be from either the Illumina Tagment DNA TDE1 Enzyme and Buffer kit or the Fapon Tnp Library Prep Kit for Illumina. You only need one kit, not both. 18. Take the 2 2?L input sample from ?20C, and mix with 30?L tagmentation mix (the same as above). 19. Put both the IP and input samples around the thermomixer to incubate at 37C for 5?min with 800?rpm shaking. There is GSK 2830371 no need to quantify the DNA concentration at this stage. Use all for the next step. The combination of S5xx and N7xx primers identifies a sample. Therefore, different samples should use different combinations of S5xx and N7xx primers. If you do not have many samples, it is recommended to use different N7xx primers, because the index in the N7xx primer is usually sequenced first on an Illumina machine. The cycle number should be chosen at the exponential phase, before reaching saturation. The shape of the size distribution of the library depends on many factors, such as the sonication and the protein of being analyzed. The majority of the DNA should fall?between 200 and 1,000?bp. We found the large fragments ( 1,000?bp) do not affect?quantification or sequencing at all. Therefore, we just leave them as they are. Asterisks indicate primer leftover, which can be removed by a further beads purification if needed. The above command is usually in one single.