title: GSM8547663: ABE8e(V106W),HEK293_site_2,rep2; Homo sapiens; OTHER
library_name: GSM8547663
library_construction_protocol: Genomic DNA was freshly extracted using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme, DC112) and stored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at −80 °C 2μL 100μM Ad1-F and 2μL 200μM Ad1-R were annealed in 1× T4 RNA ligation buffer in a thermos cycler. Similarly, hybridize 20μL Ad2-F and 20μL Ad2-R in 50mM NaCl. Afterwards, eluted DNA were incubated in a 100 ul mixture (1ul annealed Ad1-F/Ad1-R (10μM/20μM), 20% PEG-8000, 0.5mM ATP, 0.375 U/μL T4 DNA ligase high conc (NEB, M0437M) in 1× T4 RNA ligation buffer ) at 37°C for 1h and at 95°C for 2min.  The ligation products were immobilized on Streptavidin C1 beads according to the manufacturer's instruction. Free adapters were removed by washing once with 1× B&W buffer, once with 100μL stringency wash buffer (0.1% SDS, 0.1× SSC buffer) at 45°C for 3min and another once with 1× B&W buffer. To synthesize the second strand, the beads were resuspended with 48μL mixture containing 2μM Primer , 200μM each dNTP, 0.05% Tween-20 in 1× Klenow reaction buffer. After 2min at 65°C, 2μL Klenow fragment was added and the beads were incubated for 2min at 25°C and for 25min at 35°C with gentle shaking. After a second bead wash, the beads were resuspended with 100μL mixture containing 2μM each Ad2-F/Ad2-R, 5% PEG-4000, 0.05% Tween-20, 0.1U/μL T4 DNA ligase in 1×T4 DNA ligase buffer. After incubating for 1h at 22°C in a thermomixer (800 r.p.m), another bead wash was performed with B&W buffer, stringency wash buffer and B&W buffer as described above. The DNA was eluted from C1 beads using TET buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.5% Tween-20) after heating at 95°C for 1min. Eluted DNA was finally subjected to PCR amplification. Sequencing was performed by Illumina Novaseq (Azenda Life Science). Library construction was performed according to the ssDNA library construction protocol with some modifications.(PMID: 32612278)