GSM8989361: H3K27ac ChIP-seq on 1014 NOTCH2-Inactivated TRIM28-dTAG-HA  knock-in cells with DMSO Rep1; Mus musculus; ChIP-Seq
Cells were  collected, and centrifuged. The supernatant was removed and the cells were washed once in PBS. For the TRIM28-HA ChIP experiments, cells were cross-linked by incubation with 2 mM disuccinimidyl glutarate (DSG) with rocking for 30 minutes at room temperature prior to fixation step. Then, for all ChIP experiments, cells were fixed in 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 followed by rocking the cells for an additional 5 minutes at room temperature. The paraformaldehyde/glycine was then removed by spinning at 400 x g for 5 minutes at 4°C and the cells were washed twice with ice cold PBS.     The cells were then resuspended in 1 mL of lysis buffer (50 mM Hepes-NaOH pH 8, 140 mM NaCl, 1 mM 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 1 mL 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. 5% of the total lysate was removed to make genomic DNA to be used for the ChIP-sequencing input DNA. The TRIM28-HA ChIPs were performed separately also using 5% input and 95% for ChIP. 10 μg of the rabbit anti-HA (CST #3724) primary antibodies were added. For the H3K9me3 and H3K27ac ChIPs, the remaining lysate (95% of total) were equally distributed into two Eppendorf tubes to perform the H3K9me3 and H3K27ac ChIPs. 5 μg of the rabbit anti-H3k9me3 (EPR16601) (Abcam, # ab176916) or 5 μg of the rabbit anti-H3K27ac (Diagenode# C15410196) primary antibodies were added. All samples were incubated overnight while rotating at 4°C. The following morning, 50 μL 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 1 mL of low salt immune complex wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% TX-100, 2 mM 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 1 mL of high salt immune complex wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% SDS, 1% TX-100, 2 mM 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 1 mL of Lithium Chloride immune complex wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% SDS, 1% NP-40, 1 mM 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 #19101) 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 #28706) according to the manufacturer's instructions. ChIPseq libraries were prepared using IDT XGen DNAseq library prep reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer's protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on an Illumina NovaSeq X Plus targeting 40 million 150bp read pairs.