title: GSM5653828: H3K36me2_ChIP-seq_mESC_NSD1_KO; Mus musculus; ChIP-Seq
library_construction_protocol: 5 million cells per condition were collected by 300xg centrifugation and resuspended in 900µl 1X PBS at room temperature. Cells were fixed by the addition of 60µl of 16% fresh formaldehyde (1% final conc., Pierce) followed by an incubation of 10 minutes at room temperature, with rotation. The reaction was stopped by adding glycine 1M to a final concentration of 0.14M, followed by another incubation of 10 minutes at room temperature, with rotation. Cells were collected by centrifugation at 3000 rpm for 5 minutes at 4°C and washed once with 1X PBS. Aliquots of 1 million cells were flash frozen and stored at -80°C. To lyse the cells for ChIP, cells were thawed and resuspended with 1mL of lysis buffer 1 (10mM Tris-HCl pH 8.0, 0.25% Triton X-100, 10mM EDTA, 0.5mM EGTA, 1x Protease inhibitors, and 1mM PMSF), then incubated with rotation for 15 minutes at room temperature. Nuclei were pelleted by centrifugation at 1465 g for 5 minutes at 4°C. Nuclei were then resuspended with 1mL of lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 10mM EDTA, 0.5mM EGTA, 1x Protease inhibitors, and 1mM PMSF), then incubated with rotation for 10 minutes at 4°C. Nuclei were pelleted by centrifugation at 1465 g for 5 minutes at 4°C. Nuclei were then resuspended in 900uL of lysis buffer 3 (10mM Tris-HCl pH 8.0, 10mM EDTA, 0.5mM EGTA, 0.1% SDS, 1x Protease inhibitors, and 1mM PMSF) and sonicated in a Covaris M220 instrument (PIP = 75; CPB = 200; Duty Factor = 20%; Time = 10 minutes; Setpoint temperature = 7°C) using a 1mL AFA Fiber milliTube (Covaris). The sonicated lysate was centrifuged at max. speed for 10 minutes at 4°C, and the supernatant was retained.    30uL of Protein A Dynabeads (Invitrogen 10001D) were washed three times with ChIP dilution buffer (16.7mM Tris-HCl pH 8.0, 0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, and 167mM NaCl), each wash consisting of addition of 1mL of buffer and collection of the beads on a magnetic rack. The 30uL beads were then resuspended in 900uL of dilution buffer and combined with the ChIP sample to pre-clear the sample. Beads and chromatin were incubated with rotation for 2 hours at 4°C. Beads were then collected on a magnetic rack and the supernatant was retained.     2uL of SNAP-ChIP K-MetStat panel (EpiCypher, 19-1001) was added to the sample chromatin.  SNAP-ChIP K-MetStats are barcoded recombinant nucleosomes bearing distinct methyl-lysine modifications and was used as spike-in controls for ChIP reactions. 1% of the sample was saved as Input, the rest was used for ChIP. 2uL of antibody (Cell Signaling; anti-H3K27me3 (9733S), (Cell Singaling; anti-H3K36me2 (2901S)) was added to the ChIP sample. The samples were then rotated overnight at 4°C.    60uL of Protein A Dynabeads were washed three times with 1mL of ChIP dilution buffer and resuspended in 60uLof dilution buffer. Beads were then added to the ChIP sample and rotated for 2 hours at 4°C. The beads were then washed twice with Wash buffer A (50mM HEPES pH 7.9, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, and 140mM NaCl), with Wash buffer B (50mM HEPES pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, and 500mM NaCl), and with TE buffer (10mM TrisHCl pH 8.0, 1mM EDTA). Each wash consists of resuspension in 500μL buffer and rotation for 4 minutes, followed by collection of beads and removal of supernatant.     DNA was eluted in 100μL elution buffer (50mM TrisHCl pH 8.0, 1mM EDTA, and 1% SDS) at 65°C for 10 minutes shaking at 1400RPM on orbital shaker. The eluent was collected and the beads were subjected to a second round of elution with 150μL elution buffer. The ChIP eluants were pooled, and the input sample was diluted to 250μL with elution buffer. These samples were incubated at 65°C overnight to promote decrosslinking. The samples were then allowed to cool to room temperature, 15μg of RNAse A (PureLink, Invitrogen) was added, and the samples were incubated at 37°C for 30 minutes to degrade RNA. 100μg Proteinase K was then added and the samples were incubated at 56°C for two hours. DNA was purified using a MinElute PCR Purification kit (Qiagen).  DNA was sonicated again to 150bp average fragment size with a Covaris M220 instrument (PIP = 75; CPB = 200; Duty Factor = 20%; Time = 10 minutes; Setpoint temperature = 20°C) and libraries were generated using the Accel-NGS 2S Plus DNA Library Kit (Swift). Accel-NGS 2S Plus DNA Library Kit