GSM7290904: healthy human brain, antibody gH2AX, biol rep 3; Homo sapiens; OTHER
Frozen human brain tissue was thawed on ice and minced sharply into <1mm pieces. Next, 500 μl of chilled Nuclei EZ Lysis Buffer (Millipore Sigma, NUC-101, no. N3408) was added, and tissue was homogenized 10–20 times in a Dounce homogenizer. The homogenate was transferred to a 1.5-ml Eppendorf tube, and 1 ml of chilled Nuclei EZ Lysis Buffer was added. The homogenate was mixed gently with a wide-bore pipette and incubated for 5 min on ice. The homogenate was then filtered through a 70-μm mesh strainer and centrifuged at 500g for 5 min at 4 °C. Supernatant was removed, and nuclei were resuspended in 1.5 ml of Nuclei EZ Lysis Buffer and incubated for 5 min on ice. Nuclei were centrifuged at 500g for 5 min at 4 °C. After carefully removing the supernatant (pellet might be loose), nuclei were washed in wash buffer (1× PBS, 1.0% BSA and 0.2 U μl−1 of RNase Inhibitor). Nuclei were then centrifuged and resuspended in 1.4 ml of wash buffer for two additional washes. Nuclei were then filtered through a 40-μm mesh strainer. Intact nuclei were counted after counterstaining with Trypan blue in a standard cell counter. The CUT&RUN Library Prep was performed using the Illumina NovaSeq 6000 Sequencing System at the University of Kansas Medical Center – Genomics Core (Kansas City, KS). Fragmented input and immunoprecipitated chromatin (5ng) was used to initiate the TruSeq ChIP Sample Prep Kit library preparation protocol with modifications for CUT&RUN sample input (Illumina Cat# IP-202-1012).