title: GSM3898064: KDM2AB_JmjCfl_UNT_nativeinput_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: For histone H3 and H3K36me2 ChIP, 1x107 mESCs were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 125 mM glycine. The released nuclei were washed twice in PBS, then resuspended in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 and 1xPIC) and incubated on ice for 30 min. For KDM2A/B ChIP, 5x107 mESCs were resuspended in PBS and mixed with 2x106 HEK293T cells. Cells were crosslinked in 2 mM disuccinimidyl glutarate (Thermo Scientific) for 45 min at 25°C with gentle rotation, then in 1% formaldehyde for 12.5 min (methanol-free, Life Technologies). Reactions were quenched by addition of 125 mM glycine, and crosslinked cells were resuspended in lysis buffer (50mM HEPES-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX-100 and 1x PIC) and rotated for 10 min at 4°C. The released nuclei were washed (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1x PIC) for 5 min at 4°C, and the nuclear pellet resuspended in 1 ml sonication buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine and 1xPIC). For RNAPII ChIP, 5x107 mESCs were resuspended in PBS and mixed with 4x106 HEK293T cells. Cells were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 150 mM glycine, and the crosslinked cells resuspended in FA-lysis buffer for 10 min (50mM HEPES pH 7.9, 150mM NaCl, 2mM EDTA, 0.5mM EGTA, 0.5% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10mM NaF, 1mM AEBSF, 1xPIC(Roche)).  For H3, H3K36me2, KDM2A/B or RNAPII ChIP, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to approximately 0.5 kb. Sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% TritonX-100, 1mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl and 1x PIC) for H3, H3K36me2 or KDM2A/B ChIP, or in FA-lysis buffer for RNAPII ChIP. Chromatin was pre-cleared for 1 hour with either protein A agarose beads (Repligen, for H3, H3K36me2 or RNAPII ChIP) or protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 150 μg chromatin (KDM2A/B), 300 μg chromatin (RNAPII) or chromatin corresponding to 1x105 cells (H3 or H3K36me2 ChIP) was incubated overnight with the appropriate antibody: anti-H3 (in house, 15 μl), anti-H3K36me2 (in house, 15 μl), anti-KDM2A (in house, 2.4 μl), anti-KDM2B (in house, 2 μl), anti-Rbp1-NTD (CST D8L4Y, 15 μl), anti-Rbp1-CTD-Ser5P (CST D9N5I, 12.5 μl), anti-Rbp1-CTD-Ser2P (CST E1Z3G, 12.5 μl). Antibody-bound chromatin was isolated using blocked protein A agarose (H3, H3K63me2 or RNAPII ChIP) or magnetic beads (KDM2A/B ChIP) for 2 hours at 4°C. For H3, H3K36me2 or KDM2A/B ChIP, washes were performed with low salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH 8, 150mM NaCl), high salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl), LiCl buffer (250mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCl pH 8) and two washes with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). For RNAPII ChIP, washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500mM NaCl, DOC buffer (250mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 2mM EDTA, 10mM Tris-HCl pH 8) and two washes with TE buffer. ChIP DNA was eluted in elution buffer (1% SDS, 100mM NaHCO3) and crosslinks reversed overnight at 65°C with 200mM NaCl and 2 μl RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was treated identically. The following day, samples were treated with 20 μg/ml Proteinase K (Sigma) for 2 hours at 45°C and purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native ChIP-seq, 5 x 107 mouse ESCs were resuspended in 1ml RSB (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 5mM N-ethylmaleimide (NEM)), then lysed by addition of 28 ml RSB containing 0.1% NP40. The released nuclei were washed in RSC buffer (RSB supplemented with 0.25M sucrose and 3mM CaCl2), then resuspended in 1 ml RSC buffer containing  10mM NEM and 1xPIC(Roche). Each sample was then incubated with 150 units of MNase (Fermentas) at 37°C for 5 min, then 4 mM EDTA was added to halt MNase digestion. Following centrifugation at 5000 rpm for 5 min at 4°C, the supernatant (S1) was retained. The pellet was resuspended in 300 µl nucleosome release buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.2mM EDTA, 10mM NEM, 1xPIC), incubated at 4°C for 1 h, then passed five times through a 27G needle using a 1 ml syringe. After centrifugation at 5000 rpm for 5 min at 4°C, the second supernatant (S2) was combined with S1. A small aliquot of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to predominantly mono-nucleosomes. S1/S2 was diluted 10-fold in native ChIP incubation buffer (70mM NaCl, 10mM Tris-HCl pH 7.5, 2mM MgCl2, 2mM EDTA, 0.1% TritonX-100, 10mM NEM, 1xPIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with 5 μl of anti-H2AK119ub1 (CST D27C4) or anti-H3K27me3 (in-house). Antibody-bound nucleosomes were isolated by incubation for 1 hour at 4°C with protein A agarose beads, which were pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native wash buffer (20mM Tris-HCl pH 7.5, 2mM EDTA, 125mM NaCl, 0.1% TritonX-100) and once with TE buffer. ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), then purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). DNA from a matched input sample (corresponding to 10% of original ChIP reaction) was also purified.