GSM5627270: RNaseH_ssDRIP_rep2; Oryza sativa; OTHER
Genomic DNA was extracted by the phenol-chloroform method and precipitated with isopropanol. DNA fragmentation was performed at 37 °C overnight using endonucleases (Ssp I, Eco RI, Hind III, Dde I, Msp I and Rsa I; New England Biolabs, Ipswich, MA, USA); the negative control was treated with RNase H (New England Biolabs) at 37 °C overnight. DRIP was performed as described previously. The S9.6 antibody was purified from HB-8730 (ATCC, Manassas, VA, USA). The DRIPed DNA samples were sonicated to a set size of 250 bp with an M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). The sonicated DNA was denatured at 95 °C for 2 minutes and immediately placed on ice for 2 minutes to obtain ssDNA fragments. The first adapter (Adp1, GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(i7) ATCTCGTATGCCGTCTTCTGCTTG) was ligated to the 3' end of the ssDNA using Adaptase (Swift Biosciences, Ann Arbor, MI, USA) via a highly efficient, proprietary reaction that tails only the 3' ends of ssDNA and ligates the first truncated adapter to the 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequenceindependent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter (Adp2, AATGATACGGCGACCACCGAGATCTACAC(i5) ACACTCTTTCCCTACACGACGCTCTTCCGATCT) to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified. The libraries were HiSeq X Ten system.