GSM5425890: GM12878-HDAC2-100K-1; Homo sapiens; ChIP-Seq
GM12878 protocol: Cells were centrifuged at 1,600 g for 5 min and resuspended in DPBS. Our experiments usually started with 4×10^5 cells/nuclei suspended in 100 µl of DPBS. 1 µl of PIC, 1 µl of 100 mM PMSF and 100 µl lysis buffer [4% Triton X-100, 100 mM tris-HCl, 100 mM NaCl, and 30 mM MgCl2] were added, mixed by vortexing and incubated at room temperature for 10 min. 10 µl of 100 mM CaCl2 and 2.5 µl 100U MNase (88216, Thermo Fisher Scientific) were added, mixed by vortexing and incubated at room temperature for 10 min. 22 µl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The solution was centrifuged at 16,100g for 5 min at 4°C. Supernatant containing fragmented chromatin was collected into a new 1.5 ml tube and placed on ice. Chromatin volume equal to the stated number of cells per assay was taken and used for nMOWChIP, diluted to 60 µl loading volume with IP buffer if needed. Mouse protocol: A mouse brain was put on ice and PFC and cerebellum were dissected for nuclei isolation. The following steps are performed on ice and centrifugation performed at 4°C. Tissue was placed in 3 ml of ice-cold nuclei extraction buffer [0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)¬2, 0.1 mM EDTA, 10 mM tris-HCl, and 0.1% Triton X-100, with 30 µl of PIC (P8340, Sigma-Aldrich), 3 µl of 100 mM PMSF, and 3 µl of 1 M dithiothreitol added before use]. Tissue was homogenized in the grinder set (D9063, Sigma-Aldrich) by slowly douncing 15 times with pestle A and 25 times with pestle B. Homogenate was filtered through a 40 µm cell strainer into a 15 ml tube and centrifuged at 1000g for 10 min. The supernatant was removed and the pellet was resuspended in 500 µl nuclei extraction buffer and transferred to a 1.5 ml tube. 750 µl of 50% iodixanol, 7.5 µl of PIC, 0.75 µl of 100 mM PMSF and 0.75 µl of 1M dithiothreitol were added and mixed by pipetting. The mixture was centrifuged at 10,000g for 20 min and the supernatant was removed. If mixed nuclei (without separation of neurons and glia) were used for MOWChIP directly, the nuclei pellet was resuspended in 200 µl of Dulbecco's phosphate-buffered saline (DPBS). If nuclei labeling and FACS sorting were conducted, 500 µl of 2% normal goat serum (50062Z, Life Technologies) in DPBS was added to the nuclei pellet and incubated for 10 min before resuspending. Anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was diluted with DPBS to 2 ng/µl. 8 µl of anti-NeuN was added to each 500 µl of nuclei suspension and incubated at 4°C for 1 h on a rotator. The labeled nuclei were then sorted using FACS (BD FACSAria, BD Biosciences). 8 µl of non-labeled nuclei were saved as unstained control prior to addition of anti-NeuN antobody. The concentration of nuclei suspension after FACS was typically low (~1.2×10^5/ml). The nuclei were re-concentrated by adding 200 µl of 1.8 M sucrose, 5 µl of 1M CaCl2 and 3 µl of 1M Mg(Ac)2 to 1 ml of the nuclei suspension. The mixture was incubated on ice for 15 min and centrifuged at 1800g for 15 min. Supernatant was removed and the nuclei pellet was resuspended in DPBS. Nuclei were digested using the same steps as GM12878 cells. Libraries were constructed using Accel-NGS 2S Plus DNA Library kit (Swift Biosciences) following the manufacturer's instructions. 1× EvaGreen dye (Biotium) was added to the amplification mixture to monitor the PCR amplification. Library was eluted to 7 µl of low EDTA TE buffer. Library fragment size was examined using TapeStation (Agilent) and the concentration was quantified with KAPA Library Quantification kit (Kapa Biosystems). Libraries were pooled accordingly for sequencing by Illumina HiSeq 4000 SR50 mode.