GSM8787730: PRN_Adeno_M2229_Input; Mus musculus; ChIP-Seq
Adenocarcinoma and NEPC foci were identified from hematoxylin-eosin stained slides of OCT blocks of primary tumors, reviewed by pathologist. The corresponding foci in OCT blocks were cored using 2mm biopsy punch and tissues were dissociated using pestle and motor homogenizer, followed by syringes in cold PBS.  Cryosections of xenograft tumors were homogenized in a similar protocol. Dissociated cells were crosslinked in 1% methanol-free formaldehyde (Thermo Scientific, 28908) for 15 min at 37 °C. Crosslinking was quenched with 2.5 M glycine for 5 min at room temperature. Washed cell pellets were resuspended in 1 mL of lysis buffer (50 mM Tris HCl pH 8, 0.5% SDS, 10 mM EDTA with protease and phosphatase inhibitors; Thermo Fisher Scientific, 78428/78430) and lysed for 20 minutes at 4°C. Collected nuclei were resuspended in a second lysis buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, with protease and phosphatase inhibitors; Thermo Fisher Scientific, 78428/78430) and sonicated in Bioruptor Pico, Diagenode for 12mins. To the sheared chromatin, equal volumes of specific antibody and respective IgG control antibody was added and incubated overnight at 4°C. The immunoprecipitated chromatin was washed with several buffers and eluted in 300 µL of fresh elution buffer (100 mM NaHCO3 and 1% SDS). Reverse crosslinking was performed with 45 mM Tris pH 7, 170 mM NaCl at 65°C overnight, followed by RNase and proteinase K treatment and DNA was purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, 740609) as per manufacturer's recommendation. Libraries with purified immunoprecipitated DNA fragments were generated using the Hyper Prep Kit (Kapa Biosystems, KK8502). Library quality and fragment size distribution was checked in DNA High Sensitivity Bioanalyzer (Agilent, 5067-4626). Equimolar concentration of immunoprecipitated and input DNA fragments were pooled and sequenced in Illumina NovaSeq 6000 instrument at Weill Cornell Medicine Genomics core facility.