title: GSM8439497: HelaS3, mitotic, TSA, 0.025x yeast spike-in, input; Homo sapiens; ChIP-Seq
library_construction_protocol: HeLa-S3 cells were double crosslinked on plates with 2 mM DSG for 30 minutes and 1% formaldehyde for 10 minutes. Crosslinking was quenched with 1/20th volume of 2.625 M glycine and 0.5% BSA, then cells were washed 3 times with 0.5% BSA/PBS + Protease inhibitor, spinning down at 1200g for 5 minutes at 4C. Washed pellets were snap frozen then stored in -80C. Cells were lysed with LB3 lysis buffer + PIC, then chromatin was sheared on the PIXUL sonicator for 108 minutes. S2 cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine and 0.5% BSA, lysed in LB3, and sonicated on the PIXUL for 60 minutes. Yeast cultures in log phase were crosslinked with 0.86% formaldehyde for 30 minutes. The yeast cell wall was digested with Zymolase at 37°C for 60 minutes, then cells were washed 3x in PBS + PI, then resuspended in NP-S buffer + PI. Yeast were sonicated on the PIXUL for 120 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 combined then inputs were taken before immunoprecipitation for each condition, to assess the relative ratios of chromatin. To each IP, 10uL of Protein A beads and 4uL of H3K9ac, 1% Triton X-100 and 1x PIC were added. Samples were incubated overnight on rotator at 4C. IP Washes were performed as follows: 3x with 180uL of WBI, 3x with 180uL of WBIII, and 2x with 185uL TET, then eluted in 25uL of cold TT Buffer. Inputs were incubated with Speedbeads, in 12% PEG and 1M NaCl for 10 minutes at room temperature, then washed twice with 80% EtOH, and eluted in 25uL cold TT Buffer. From inputs and IPs, libraries were prepared with the NEB Ultra II Library Prep Kit. Barcoded adapters were from BioO NEXTflex ChIP-seq Adapters. Libraries were decrosslinked and cleaned with Speedbeads, then PCR amplified with Solexa 1GA and 1GB primers, and size selected with a final Speedbead cleanup. Library size was estimated with agarose gel, concentrations measured with Qubit HS DNA kit.