GSM7908397: Rloop_N_2; Homo sapiens; OTHER
The purified genomic DNA was subsequently mixed with 5 μl of 20 mM dibenzocyclooctyne-PEG4-biotin conjugate (Sigma-Aldrich, 760749) and 10 μl of 10× phosphate-buffered saline (PBS), and the total volume was adjusted to 100 μl with 25 mM K3BO3. The resulting mixture was shaken gently at 37°C for 1.5 h, followed by addition of 5 μl of 10 mg/mL RNase A (Thermo Fisher Scientific, 12091039), and the mixture was shaken at 37°C for another 5 min. After the reaction, the DNA was isolated using the DNA Clean & Concentrator-5 kit (Zymo Research, D4013) and fragmented by sonication to 150 to 350 base pairs (bp). The sonicated DNA was employed for enrichment using 10 μl of Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific, 65001). The beads were washed with 1× binding and washing (B&W) buffer (5 mM tris-HCl, pH 7.4, 0.5 mM EDTA, 1 M NaCl, and 0.05% Tween 20), resuspended in 95 μl of 2× B&W buffer, and mixed with the sonicated DNA. The suspension was incubated at room temperature for 15 min. The beads were then washed once with 1× B&W buffer, twice with 100 mM NaOH solution to denature the dsDNA and remove the DNA strands that are not labeled by N3-kethoxal, and once again with 1× B&W buffer. The DNA was eluted from the beads by heating the beads in 10 μl H2O at 95°C for 10 min. The ensuing enriched DNA and the corresponding input were employed for library construction using the Accel-NGS Methyl-Seq DNA Library Kit (Swift, 30024) following the manufacturer's instructions. Subsequently, the purified DNA libraries were assessed using an Agilent 2100 Bioanalyzer and multiplexed for sequencing on a MGISEQ-2000 platform (BGI). PROTOCOL: Strand-specific kethoxal-assisted single-stranded DNA (ssDNA) sequencing (spKAS-seq)