GSM1216734: chr11-1 chr17-1; Arabidopsis thaliana; MNase-Seq
For nuclear extraction, two-week-old Arabidopsis seedlings without roots were ground to ﬁne powder in liquid nitrogen and resuspended in the lysis buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 10 mM β-mercaptoethanol and Protease Inhibitor Cocktail, Roche). After lysis for about half an hour, the lysis mixture were filtered through 40 μm cell strainer (BD). The filtered plant extracts were centrifuged for 20 min at 3,200 g to obtain the nuclei pellet. Isolated nuclei were washed each time with HBB buffer (25 mM Tris-Cl pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X and 10 mM β-mercaptoethanol) and HBC buffer (25 mM Tris-Cl pH 7.6, 25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM β-mercaptoethanol and 20% glycerol). The nuclei were resuspended with 100 μl HBC buffer and flashed frozen in liquid nitrogen. (1) Perform End Repair. Prepare the following reaction mix: 85 μl of Eluted DNA and 5 μl of Enzyme mixture. Incubate in the thermal cycler for 30 minutes at 20 °C. Purify production with qiaquick or minelute. (2) Add “A” Bases to the 3' End of the DNA Fragments. Prepare the following reaction mix: 32 μl of DNA sample, 5 μl of NEBbuffer2, 10 μl of 1 mM dATP, 3 μl of Klenow exo (3’ to 5’ exo minus). The total volume should be 50 μl. Incubate for 30 minutes at 37 °C. Purify production with qiaquick or minelute. (3) Ligate Adapters to DNA Fragments. Prepare the following reaction mix: 37 μl of DNA sample + H2O, 5 μl of DNA ligase buffer, 3 μl of Adapter oligo mix, 5 μl of DNA ligase. The total volume should be 50 μl. Incubate for 60 minutes at 16 °C. Use a QIAGEN Qiaquick Kit (QIAGEN, part # 28104) to purify the DNA. (4) Enrich the Adapter-Modified DNA Fragments by PCR. Prepare the following PCR reaction mix: 36.5 μl of DNA sample + H2O, 10 μl of 5× Phusion buffer, 0.5 μl of Phusion*DNA polymerase, 1.25 μl of PCR primer, 1.25 μl of PCR primer 2.1, total 0.5 μl of 100 mM dNTP. The total volume should be 50 μl. Amplify using the following PCR protocol: 1 min at 98 °C, [10 seconds at 98 °C, 30 seconds at 60 °C, 30 seconds at 72 °C] for N cycles (dependent on the initial amount of DNA), 5 minutes at 72 °C, hold at 4 °C. Use a QIAGEN Qiaquick Kit (QIAGEN, part # 28104) to purify the DNA. Library construction was performed by Genergy Biotechnology (Shanghai) Co. LTD.
GEO
GSM1216734