GSM8694744: 4acC_mDIP_Zeamay_B73_Rep1; Zea mays; ChIP-Seq
Arabidopsis seedings and the leaf of rice were ground in liquid nitrogen, gDNA of Escherichia coli,Arabidopsis,rice were lysed with 10%SDS (final concentration: 0.5%) and proteinase K (final concentration: 0.1 mg/ml) and 1×TE buffe at 37°C with shaking at 180~200 rpm for 6 hours. At the end of the incubation, 1/4 volume of 5 M KAc was added, mixed thoroughly and placed on ice for 10~20 minutes. Add 1 volume of phenol: chloroform: isoamyl alcohol (25:24:1) and vortex to mix well, then centrifuge at 12000 × g for 10 min at 4°C. Transfer the supernatant to a new EP tube, add 1 volume of chloroform: isoamyl alcohol (24:1) and mix well by vortexing. Centrifuge at 12,000 × g for 10 min at 4°C. Transfer 90% of the supernatant to a new EP tube. One volume of isopropanol was added to the supernatant and precipitated at -80°C for 1 hour. The DNA precipitate was then washed twice with 75% anhydrous ethanol, and later dried to a dry powder and stored in a -20°C refrigerator for subsequent experiments. Maize and soybean genomic gDNA was extracted using Plant Genomic DNA Extraction Kit. (Tiangen, Stock No. DP-305) For mDlP-seq library construction, 1~5 ug of gDNA was digested overnight with RNase A(EN0531) and RNase H(M0297S) and then purified by the phenol-chloroform extraction method followed by resuspending the pellet in 130 ul low EDTA TE, purified gDNA was sonicated into 200-400 bp fragments with Covaris ME220 (Covaris,70 W, 20% duty factor, 1,000 cycles per burst, 130 seconds, at 4℃). The sonicated DNA was denatured at 95℃ for 10 minutes and immediately chilled on ice for 10 minutes, and then barcoded in the tailing for each sample at 37℃ for 1 hour and 95℃ for 10 minutes. The reaction system contained 1× T4 DNA ligase buffer, 10% (w/v) PEG 8000, 2 μl of barcoding adapter/splint mixture (20 μM/40 μM), 0.5 mM dGTP, 20 units of TdT, 30 units of T4 PNK, 30 units of T4 DNA ligase, and 200 ng sample gDNA. After the reaction, 1 μl of 0.5 M EDTA(pH=8.0) was added to each tube to stop the reaction. Then barcoded samples were collected into one tube and purified by the phenol-chloroform. Ten percent of the purified DNA was saved as input, the remaining DNA was incubated overnight at 4°C while gently inverting on a rotisserie shaker in 500 ul buffer with antibodies and 50 μl of 10× DIP buffer (100 mM NaPO4 (pH 7.0), 1.4 M NaCl, 0.5% Triton X-100). The ratios of antibody/gDNA are dependent on the number of barcoded samples. Next day, 30 μl of Dynabeads™ Protein A/G beads (Thermo Fisher Scientific: 26162) was added and incubated at 4°C for 5 hours. Then, the beads were washed 5 times (5 minutes each time) with 1 ml 1× DIP buffer at room temperature. After DIP, DNA modifications on Protein A/G beads were released by heating at 95 °C for 3 min and extended at 63 °C for 5 min in an 60 µl reaction containing 1 × KAPA HiFi HotStart ReadyMix and 0.25 µM P7TruSeqHTRev to produce dsDNA followed by purification with SPRIselect™ beads (Beckman Coulter) according to manufacturer's instructions (1.2 × volumes). Then, the purified dsDNA was ligated to the second adapter in a 40 µl reaction (1× T4 DNA ligase buffer, 0.5 µM P5TruSeqHT/P5SPlint mixture (1:1), and 10 Weiss units of T4 DNA ligase) at 25 °C for 1 hour and purified with SPRIselect™ beads (1.0 × volumes). Lastly, adapters ligated DNA was amplified for 10-20 cycles that were determined by qPCR (20 s at 98 °C denature, 1 min at 52 °C annealing, and 1 min at 72 °C extension) with indexing amplification primers in a 24 µl reaction (0.5 µM P5 index primer, 0.5 µM P7 index primer, and 1× KAPA HiFi HotStart ReadyMix) and purified twice with SPRIselect™ beads (0.85 × volumes)