GSM6416158: 293T-VEGFA-sgRNADel-PD_rep2; Homo sapiens; OTHER
Genomic DNA was freshly extracted using CWBIO universal genomic DNA kit (CWbiotech, CW2298M) and stored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at -80 °C. Extracted genomic DNA was fragmented into ~300 bp through Covaris Focused-ultrasonicator™ Instrument (ME220), followed by a purification using DNA Clean & Concentrator-5 (Vistech, DC2005). ~5 ug DNA fragments and 10 pg spike-in model sequences were subjected to end repair with NEBNext End Repair Module (NEB, E6050), and 1 μl E. coli ligase (NEB, M0205) was added to repair nicks in DNA. Then hydroxylamine protection of endogenous 5fC was performed in 100 mM MES buffer (pH 5.0), 10 mM O-ethylhydroxylamine (EtONH2, Aldrich, 274992) at 37 °C for 6 h. The product underwent 5fC protection was diluted 2.5 times and purified with DNA Clean & Concentrator-5. Next, dA-tailing procedure was carried out using NEBNext dA-Tailing Module (NEB, E6053) followed by a DNA purification with 1.8 × AMPure XP beads. DNA damage that may interfere the subsequent labeling step was repaired in a mixture of 2 μl endonuclease IV (Endo IV, NEB, M0304), 1 μl Bst full-length polymerase (NEB, M0328), 2 μl Taq ligase (NEB, M0208), 1 μl NAD+ (NEB, B9007), 1 μl dNTP (2.5 mM each) in NEBuffer 3 for 1 h at 37 °C and 1 h at 45 °C. DNA was purified and subjected to in vitro BER labeling reaction containing a mixture of 1 μl UDG (NEB, M0280), 1.5 μl Endo IV, 0.8 μl Bst full-length polymerase, 1.7 μl Taq ligase, 1 μl NAD+, 200 nM biotin-dUTP, 800 nM d5fCTP, 200 nM dATP and 200 nM dGTP in NEBuffer 3 for 40 min at 37 °C. The DNA was incubated with 75 mM of malononitrile in 10 mM Tris-HCl (pH 7.0) at 37°C for 20 h in an enclosed thermomixer (Eppendorf, 850 rpm), away from light. The reaction product was then purified with 1.8 × AMPure XP beads. Labeled fragments were enriched by streptavidin C1 beads (Invitrogen) following the manufacturer's instruction. Y adaptor (see Supplementary Note 1) was ligated (NEBNext Quick Ligation Module, E6056) to double-stranded DNA on streptavidin C1 beads so that free adaptor can be removed by three-times washing of 1×B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). After washing once with 1× SSC buffer (pH 7.0), beads with biotinylated DNA strands were resuspended in freshly prepared 0.15 M NaOH and incubated at room temperature for 10 min, followed by a sequential washing of 1× SSC buffer (7.0) and 1× TE buffer (pH 7.5) for once. The DNA was eluted from C1 beads using deionized water after heating at 95 °C for 3 min. Eluted DNA was finally subjected to library amplification with indexed primers (NEB). To obtain sufficient DNA template, 2 rounds of amplification were first performed by using MightyAmp DNA polymerase (Takara), which was proved to read through regions containing biotin-dU and d5fC-malononitrile sites more easily. The PCR product was recovered via DNA Clean & Concentrator-5 and then PCR amplified for ~12 cycles using NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB, M0543). The constructed libraries were checked using the Agilent 2100 bioanalyzer. Sequencing was performed by Illumina Hiseq X Ten.