GSM9700295: mESC-RBBP5-FKBP, auxin 0hr, biological replicate 1, input, tech rep 2; Mus musculus; ChIP-Seq
HCT116 or HeLa-S3 cells were double crosslinked on plates with 2 mM DSG for 30 minutes (Fisher Scientific NC1736108) and 1% formaldehyde (Pierce ThermoFisher PI28906) for 10 minutes. Crosslinking was quenched with 1/20th volume of 2.625 M glycine (EMD Millipore 357002) and 0.5% BSA (NEB B9000S). Cells were washed by resuspending 3 times with 0.5% BSA/PBS + Protease inhibitor (PIC, Roche 11697498001), spinning down at 1200g for 5 minutes at 4°C and removing the supernatant. Washed pellets were snap frozen in dry ice then stored in -80°C. Cells were lysed with LB3 lysis buffer + PIC, then chromatin was sheared on the PIXUL sonicator (Active Motif 53130) for 108 minutes. S2 cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine and 10% BSA to a final concentration of 0.5%, lysed in LB3 buffer, and sonicated on the PIXUL for 60 minutes. S. cerevisiae cultures in log phase were crosslinked with 0.86% formaldehyde for 30 minutes on a room temperature rotating shaker. The cell wall was digested with Zymolase at 37°C for 60 minutes, cells were washed by pelleting 3x at 5000g and washing in PBS + Protease inhibitor, then resuspended in NP-S buffer + PI. Sheared S. cerevisiae chromatin was sonicated on the PIXUL for 108 minutes. All PIXUL sonication was done with settings Pulse (N) = 50, PRF (kHz) = 1.00, Burst Rate (Hz) = 20, only Process Time was varied. Sheared chromatin from each species were clarified by centrifugation at 15000g, for 10 minutes at 4°C, then combined and inputs were taken before immunoprecipitation for each condition, to assess the relative ratios of chromatin. To HeLa-S3 cells, approximately 100k S2 and 500k S. cerevisiae cells were added per immunoprecipitation reaction. To HCT116 cells, approximately 500k S2 and 100k S. cerevisiae cells were added to each immunoprecipitation reaction. To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction. To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction. Below are the volumes per IP of each antibody used: CST H3K27ac (D5E4): 1uL CST H3K4me3 (C42D8): 1uL CST H3K4me1 (D1A9): 1uL CST H3K36me3 (D5A7): 1uL CST Rpb1 NTD (D8L4Y): 4uL CST H3K9ac (C5B11): 4uL Samples were incubated overnight on a rotator at 4°C. IP Washes were performed: 3x with 180uL of WBI, 3x with 180uL of WBIII, and 2x with 185uL TET, then eluted in 25uL of cold TT Buffer. Input chromatin containing target and spike-in species' chromatin from each biological condition were diluted 1:62.5x in TT buffer to a final volume of 25uL. Libraries were prepared with the NEB Ultra II Library Prep Kit (NEB E7645L). NEXTflex Dual-Barcode ChIP-seq Adapters (NOVA-514151) were added at 0.25uM or 0.5uM or 0.625uM. Libraries were then de-crosslinked at 55°C for 1hr, then 65°C overnight and cleaned with Speedbeads, 8.6% PEG and 0.8M NaCl and eluted in TT buffer. Next libraries were PCR amplified with 1uM Solexa 1GA and 1GB primers and NEB Ultra II Q5 Mastermix (PCR protocol: 98C for 30'', then 12 or 13 cycles of 98C for 10'', 60C for 15'', 72C for 30'', then hold at 72C for 2'). A final cleanup was done with Speedbeads, 8.5% PEG and 1M NaCl, washed twice with 80% EtOH, then the final libraries were eluted in 20uL TT. Library size was estimated by running 6uL of eluted library on a 1.8% agarose gel and concentration was quantified with Qubit HS dsDNA kit (Invitrogen Q32854). Libraries were pooled and sequenced on Illumina NextSeq 550 or NextSeq 2000.