GSM5966604
Genomic DNA was isolated from mouse heart tissue with Gentra Puregene Tissue Kit without RNase A treatment. HydEn-seq was performed by hydrolysis of 1 mg of genomic DNA with 0.3 M KOH for 2 h at 55°C. After hydrolysis DNA was denatured for 3 min at 85°C, cooled on ice and treated with 10 U T4 Polynucleotide Kinase (3´ phosphatase minus) in 1 mM ATP for 20 min at 37°C and then treated for 20 min at 65°C. DNA was purified using 1.8 volumes of CleanNGS beads and eluted in 14 ml EB. 13 ml DNA was denatured for 3 min at 85 °C and ligated to 1 mM ARC140 using 10 U T4 RNA ligase in 25% PEG3350, 1 mM CoCl3(NH3)6 and 20 mM ATP overnight at room temperature. The day after, two washes was performed using 0.8 volumes of CleanNGS beads and eluted with 20 ml EB in the first step and 13.8 ml EB in the second step. DNA was denatured for 3 min at 85°C and incubated for 5 min with 1xBF for T7 DNA polymerase, 0.2 mM dNTPs, 40 mg/ml BSA, and 1 mM of the adapter consisting of ARC76/ARC77. Reaction was then treated with 4 U of T7 DNA polymerase for 5 min. Product was purified with 0.8 volumes of CleanNGS beads and amplified using primers ARC78-ARC84 and ARC160. All oligos are from IDT. Sequences are provided in table S1. * indicates a phosphorothioate bond. ARC140 contains a 5´-amino group instead of a 5´-OH group, in combination with a C6 linker.