GSM3290194: C7_Mta2_Rep2; Mus musculus; ChIP-Seq
For crosslinking-ChIP, 40-45 million ES cells were resuspended in 10ml PBS with 2 mM EGS for 45 min, followed by 1% formaldehyde for a further 10 minutes. The reactions were quenched by the addition of glycine to a final concentration of 125 mM. Cells were first lysed for 10 minutes on ice in LB1 (50 mM Hepes pH 7.9, 140 mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton). The nuclei was pelleted and washed for 5 minutes in LB2 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and pelleted again. The nuclei was suspended in 1 ml of LB3 (10 mM Tris pH 8.0, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a BioRuptor sonicator (Diagenode) with for 25 cycles 30 sec on and 30 sec off, followed by centrifugation at maximum speed for 15 min at 4 ºC, and either used freshly or snap freeze and stored at -80 ºC. For calibrated ChIP-seq of HDAC1, MTA2, Pol II Ser5P, and Pol II Ser2P 40 million ES cells and 10 million Drosophila SG4 Cells were pooled in PBS prior to crosslinking. For each IP, 100 µl of chromatin was diluted with ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl) prior to pre-clearing with protein A agarose beads or magnetic Dynabeads (Invitrogen) blocked with 1 mg/mL BSA and 1 mg/mL yeast tRNA at 4 ºC for 1 h. Precleared chromatin samples were further incubated overnight with relevant antibodies at 4 ºC with rotation. For each calibrated ChIP sample, 5-8 μg antibody plus 2 μg Drosophila-specific H2Av antibody (Active Motif) were used. Antibody-bound chromatin was isolated on blocked protein A beads, and followed by washes with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer ((0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)), and twice with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) buffer. The DNA was eluted with 1% SDS and 100 mM NaHCO3, and reversed crosslinked at 65 ºC overnight in the presence of 200mM NaCl, following by treatment with RNaseA and Proteinase K. DNA was purified by ChIP DNA Clean and Concentrator kit (Zymo) and quantified by dsDNA Qubit reagents. For calibrated native ChIP (H3K27ac and H3K9ac), 40 million ES cells and 10 million Drosophila SG4 Cells were pooled and lysed in RSB (10 mM Tris pH 8, 10 mM NaCl, 3 mM MgCl2) + 0.1% NP40 for 5 min on ice with gentle inversion. Nuclei was resuspended in 1 ml of RSB + 0.25 M sucrose + 3mM CaCl2, treated with 200U of MNase (Fermentas) for 5 minutes at 37ºC, quenched with 4 µl of 1M EDTA, then centrifuged at 5000 rpm for 5 minutes. The supernatant was transferred to a fresh tube as fraction S1. The chromatin pellet was incubated for 1 h in 300 µl of nucleosome release buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.2 mM EDTA), carefully passed 5 times through a 27G needle using a 1 ml syringe, then centrifuged at 5000 rpm for 5 minutes. The supernatant from this S2 fraction was combined with S1. For each ChIP reaction, 80 µl of chromatin was diluted in Native ChIP incubation buffer (10 mM Tris pH 7.5, 70 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton) to 1 ml and incubated with antibody overnight at 4ºC. Samples were incubated with 50 µl protein A agarose beads preblocked in Native ChIP incubation buffer with 1 mg/ml BSA and 1 mg/ml yeast tRNA, then washed for a total of 4 times in Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton) and once in TE. The DNA was eluted with 1% SDS and 100 mM NaHCO3, and was purified using ChIP DNA Clean and Concentrator kit (Zymo). Approximately 5-20 ng of ChIPed DNA were used for library prep using NEBNext Ultra II DNA Library Prep Kit with NEBNext Single index, and then further quantified by qPCR with KAPA Library Quantification DNA standards (KAPA Biosystems) and SensiMix SYBR (Bioline, UK). The libraries were pooled and 2 ÃƒÂ— 42 paired end sequencing was performed on the Illumina NextSeq500.