GSM6454712: InputH3K4me2_H3K27me3_sgCTRL_1014; Mus musculus; ChIP-Seq
KDM6A ChIP sequencing experiments were performed by first cross-linking cells with 2 mM disuccinimidyl glutarate (DSG) with rocking for 30 minutes at room temperature prior to fixation with 1% paraformaldehyde diluted in PBS for 10 minutes at room temperature. H3K4me2 and H3K27me3 ChIP was performed by fixation with 1% paraformaldehyde diluted in PBS for 10 minutes at room temperature. Excess formaldehyde was quenched by dropwise addition of freshly made Glycine at a final concentration 1.25 M. The cells were then resuspended in 1 mL of lysis buffer (50mM Hepes-NaOH pH 8, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100 supplemented with a protease inhibitors and phosphatase inhibitors) and incubated by rotating for 10 minutes at 4°C. Intact nuclei were then collected by centrifugation at 1900 x g for 5 minutes at 4°C. The supernatant was then gently aspirated and the pellet was resuspended in 1 mL of wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, supplemented with a protease inhibitor cocktail and phosphatase inhibitors) and incubated by rotating for 10 minutes at 4°C. The nuclei were again collected by centrifugation at 1900 x g for 5 minutes at 4°C. The supernatant was then gently aspirated and the tube was rinsed with 500 µL of shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8.0 supplemented with protease inhibitors and phosphatase inhibitors). The nuclei were again collected by centrifugation at 1900 x g for 5 minutes at 4°C, the supernatant was carefully removed, and the pellet was gently resuspended in 1mL shearing buffer and transferred to a 1mL AFA fiber tube (Covaris #520130). The chromatin was then sonicated using a Covaris E220 sonicator at 100 peak incident power, 10% duty cycles, 200 cycles per burst, water level 15 (left indicator)/10 (right indicator) for 900 seconds. The samples were then transferred to 1.5 mL Eppendorf tubes and centrifuged in an Eppendorf microcentrifuge at 15,900 x g for 5 minutes at 4°C. 900 µL of the supernatants were transferred to a new Eppendorf tube, to which 1% Triton X-100 and 150 mM NaCl were added. 20µl of Dynabeads Protein G (ThermoFisher #10004D), prewashed with Shearing Buffer, was then added and samples were rotated for 1 hour at 4°C to pre-clear the sonicated chromatin. The Dynabeads were then removed using a magnetic stand and the supernatants were transferred to new tubes. 2.5% of the total lysate was removed to make genomic DNA to be used for the ChIP-sequencing input DNA. For the H3K27me3 and H3K4me2 ChIP, the remaining lysate (95% of total) were equally distributed into two Eppendorf tubes to perform the H3K27me3 and H3K4me2 ChIPs. 3 µg of the rabbit anti-H3k27me3 (C36B11) (Cell signaling, #9733) or 3 µg of the rabbit anti-H3K4me2 (Abcam #Ab32356) primary antibodies were added. For the KDM6A ChIP, the remaining 95% were incubated with 10 µg of the rabbit anti-KDM6A [Bethyl lab, A302-374A (UTX antibody)]. Same amount of rabbit IgG isotype control (Cell Signaling #3900) with the same amount of lysate was used as a control for each ChIP. All samples were incubated overnight while rotating at 4°C. The following morning, 50µL of Dynabeads, prewashed with Shearing buffer, were added and the samples were incubated for another 2 hours while rotating at 4°C. A magnetic stand was used to isolate the Dynabeads.  The supernatant was removed and the Dynabeads were washed once in 1mL of low salt immune complex wash buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 0.1% SDS, 1% TX-100, 2mM EDTA) and incubated for 5 minutes while rotating at 4°C.  The beads were again isolated using a magnetic stand.  Following removal of the supernatant the Dynabeads were washed once in 1mL of high salt immune complex wash buffer (20mM Tris-HCl pH 8.0, 500mM NaCl, 0.1% SDS, 1% TX-100, 2mM EDTA) and incubated for 5 minutes while rotating at 4°C.  The beads were again pelleted with a magnetic stand.  Following removal of the supernatant, the Dynabeads were washed once in 1mL of Lithium Chloride immune complex wash buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% SDS, 1% NP-40, 1mM EDTA) and incubated for 5 minutes while rotating at 4°C. The beads were then washed twice in TE pH8 at room temperature and the beads were then resuspended in 100µL TE pH8. RNase A (Qiagen) was then added to a final concentration of 0.2 mg/mL and the samples were incubated for 30 minutes at 37°C. To reverse the formaldehyde crosslinking and remove protein, proteinase K was then added to a final concentration of 0.8 mg/mL and the samples were incubated for 30 minutes at 42°C and then an additional 6 hours at 65°C. 300 µL QG buffer and 100 µL of isopropanol was then added to each sample and a magnetic stand was used to pellet the Dynabeads. The supernatant was then collected and DNA was isolated using QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. DNA concentration was determined using the Nanodrop 8000 (Thermofisher Scientific) and sample were stored at -80°C.