GSM9450703: H4K5Ac G217B A; Histoplasma ohiense; ChIP-Seq
Cultures processed for ChIP-seq were crosslinked with formaldehyde (Polysciences 18814-10) added to a final concentration of 1 % and kept under culture conditions (shaking at 37 °C with 5 % CO2 or ambient RT) for 20 minutes to allow fixing to take place. Crosslinking was quenched with glycine added to a final concentration of 125 mM for 5 minutes at RT. Fixed cells were pelleted at 3000 g for 10 minutes at 4 °C and washed twice with TBS (20 mM Tris-HCl (pH 7.5), 150 mM NaCl). Washed cells were harvested through filtration, aliquoted into pellets of equal mass (between 150-300 mg/tube) and flash frozen in liquid nitrogen as described above. Frozen pellets were resuspended in lysis buffer (50 mM Hepes/KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % sodium deoxycholate, protease/phosphatase inhibitors) at 4 °C before being bead beat with 500 µL of 0.5 mm zirconia beads in 5 x 1 minute cycles at RT with 2 minute rests on ice in between. The resulting lysate was then spun down at 8000 rpm for 10 minutes at 4 °C to isolate the insoluble chromatin fraction. This chromatin pellet was resuspended in 600-900 µL of lysis buffer and sonicated at 4 °C using the Diagenode Bioruptor® Pico for 30-35 cycles (30s on, 30s off). Sonicated samples were spun down for 5 min at 14,000 rpm at 4 °C to pellet cell debris and aliquots for input DNA were reserved at -20 °C in TE + 1 % SDS. Input samples were also used to assess shearing using a High Sensitivity DNA Bioanalyzer chip from Agilent. Equal lysate volumes were aliquoted for each IP to be performed and brought to a final volume of 500 µL with lysis buffer and 20 µg of each polyclonal ⍺-Ryp antibody (Ryp1: 3873, Ryp2: 387, and Ryp3: 356) or 3 µg of a ChIP-grade monoclonal ⍺-histone-acetyl antibody (H3K9Ac: ab177177 , H3K14Ac: ab52946, H4K16Ac: ab109463). Samples were incubated overnight with agitation at 4 °C before being incubated with 50 µL of a 50% slurry of Protein A Dynabeads (Life Technologies Corporation 10002D) pre-washed 2x with TBS and 3x lysis buffer for 2 hours at 4 °C with agitation.  Immunoprecipitated DNA was recovered through pulldown on Protein A beads using a magnetic rack. Pellets containing DNA-protein complexes were washed 2x in lysis buffer, 2x with high-salt (500 mM NaCl) lysis buffer, 2x with wash buffer (10 mM Tris/HCl (pH 8.0), 250 mM LiCl, 0.5 % NP-40, 0.5 % Na-deoxycholate, 1 mM EDTA) and 1x with TE before adding elution buffer (50 mM Tris/HCl (pH 8.0), 10 mM EDTA, 1 % SDS) and incubating at 65 °C for 10 minutes. DNA was eluted using a magnetic rack and the resulting beads were subjected to second round of resuspension and elution with 150 µl TE + 0.65 % SDS and pooled with the initial eluate to maximize total yield of DNA. IP and input samples were treated with 1 µL of Proteinase K (20 mg/mL Qiagen 19131) and incubated at 65 °C for approximately 16 hours. IP and Input DNA samples were then treated with 2 µg of RNase A (Qiagen) and incubated at 37 °C for 1 hour. DNA was then purified using the Zymo ChIP DNA Clean & Concentrator Kit (D5205) and eluted in 31 µL of ddH2O or TE. Purified IP and input DNA were used for Illumina library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB E7103S) and Illumina dual-indexed primer pairs sourced from the UCSF CAT. The quality and size distribution of each library was assessed using the Agilent High Sensitivity DNA Bioanalyzer Kit (Agilent Technologies 5067-4626) to ensure proper fragment size and the absence of contaminating adapters. Final library concentrations were measured using a Qubit HS dsDNA assay kit (Life Technologies Q33230) and used to pool equimolar amounts of each sample into final libraries for sequencing. Pooled libraries were used for paired-end sequencing using one NovaSeq X 10B lane at the UCSF CAT.