Protease k

Only epithelial cells confirmed by transcriptome analysis were further selected for DNA library construction. We used 5 µL of protein lysis buffer containing 2.5 µL of M-digestion buffer (Zymo, Cat# D5044) and 0.5 µL of protease K (NEB, Cat# P8107S) to resuspend the bead-trapped nuclei. The genomic DNA in each cell was released after incubation at 50 °C for 1 h. Then, the genomic DNA lysates were stored at −80 °C, and we selected the genomic DNA of cells that were classified as epithelial cells through transcriptome analysis to perform DNA amplification. In brief, bisulfite conversion was carried out using the EZ-96 DNA Methylation-Direct™ Mag Prep Kit (Zymo, Cat# D5044). Specifically, we added only 32.5 µL of CT conversion reagent to 5 µL of single-cell genomic DNA lysate. We followed the steps of the single-cell whole-genome bisulfite sequencing workflow^{40 (link)} with minor modifications, including (1) We used random primers containing N6 sequences. (2) We performed a total of 4 rounds of olig1 tagging and skipped the Exo I digestion; instead, we removed the free primers by purification with 0.8 volume of Ampure XP beads (Beckman, Cat# A63882). Finally, we performed 16 cycles of the indexing PCR program at 98 °C for 15 s, 65 °C for 30 s, and 72 °C for 1 min. The DNA library for each cell was sequenced for 2 Gb (~0.6×) on the Illumina HiSeq 4000 platform.

Patient saliva samples were previously collected for COVID-19 diagnosis at a UIC clinical lab. Patient inclusion criteria: adults of age 18 years or older; exclusion criteria: children. In addition, we also analyzed pooled saliva samples from healthy individuals (Innovative Research). Saliva samples (50-100 µL) were first diluted with 0.7X volume of TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). Then, 1X RNAsecure (25X, ThermoFisher) and 50 units/mL protease K (800 units/ml, NEB) were added to make 1:1 dilution of the saliva sample. The reaction mixture was incubated at room temperature (20-25°C) for 10 min, 65°C for 10 min, and finally 100°C for 12 min to inactivate protease K. For spike-in experiments, heat-inactivated SAR-CoV-2 particles were added to the saliva before TE dilution. For patient saliva samples (n=20), they were first heated at 65°C for 30 min to inactivate the SARS-CoV-2 virus, and then further processed as described above. For each saliva sample, an aliquot (300 µL) was used for RNA isolation with the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (A42352, Applied Biosystems), followed by RT-qPCR.

The constructed transpososome was used to tagment about 3 femtograms of yeast genomic DNA (ATCC Cat. No. 9763), which was serially diluted from a stock of 6 ng/μL, to generate a primary library^{13 (link)}. The transposase was removed from DNA by Protease K (New England Biolabs, Cat. No. P8107S) treatment and then Protease K was denatured by heat. The primary library was sequentially amplified by Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific Cat. No. F565L) with NEX8a for three cycles, and then together with Illumina Read1 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′) and Read2 (5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′) primer pair for 20 cycles, both with 30-min extension time at 65 °C. The amplified library was cleaned with AMPure (Beckman Coulter Cat. No. A63882) beads twice and eluted in 20 μL 10 Tris buffer, 8 μL of the eluents was taken to be barcoded in a 20 μL reaction in 6 cycles. The barcoded libraries were cleaned with AMPure beads twice and loaded onto MiSeq (Illumina, Cat. No. MS-102-3001) for sequencing 80 bases for Read1 and 70 bases for Read2, after quantitation following the manufacturer’s protocol^{14 (link)}.

Cells were plated and treated with DOX at a concentration of 0.2 µg/mL or equivalent volume of vehicle for 24 h. Cells were washed in cold PBS, scraped, and then lysed with a buffer containing 0.5% Nonidet, 0.5 mM DTT, 20 mM Tris-HCL pH7.5, 150 mM KCL, 2 mM EDTA, 1 mM NaF, and inhibitors of RNases, proteases, and phosphatases. Ten percent of total lysate was removed and kept as the input samples and the remainder used for immunoprecipitation. Ten micrograms of anti-AGO2 (11A9, SAB4200085, Sigma-Aldrich) or anti-IgG (Sigma-Aldrich) antibodies were bound to sepharose beads in the presence of heparin. Precleared lysates were then incubated with the appropriate antibody-bound beads, and the immunoprecipitated proteins were then washed and incubated with DNase I in the presence of DNase buffer (Promega) followed by protease K (New England Biolabs) in the presence of 2× protease buffer (New England Biolabs). RNA extraction was then performed using phenol chloroform separation and ethanol/sodium acetate precipitation. RNA pellets were washed in ethanol and quantified using a Nanodrop.

Uvr proteins were purified as previously described [27 ]. Monoclonal antibodies to acrolein-dG DNA adducts were prepared as described [14 (link)]. UPIII antibodies were purchased from Abcam (Cambridge, MA), α-tubulin from Calbiochem (Billerica, MA), and antibodies against mouse/rabbit IgG from Amersham Biosciences (Pittsburgh, PA). Normal human bladder epithelial cells (HBEP cells) were obtained from Zen-Bio (Research Triangle Park, NC). UROtsa cells were obtained from the laboratory of JR Masters (University College London, UK) and normal human fibroblasts (CCL202 cells) were obtained from ATCC (American Type Culture Collection, Manassas, VA). Plasmid pSP189 was prepared as described by Canella and Seidman [28 (link)]. T4 kinase, protease K, and RNase A were purchased from New England Biolabs (Ipswich, MA); γ-³²P-ATP and α-³²P-dATP were purchased from Perkin Elmer (Waltham, MA), and nuclease P1 and phosphodiesterase II were purchased from Sigma (St. Louis, MO) and Worthington (Lakewood, NJ), respectively. Acrolein (purity 90%) was purchased from Sigma and N-hydroxy-4-ABP (N-OH-4-ABP) was synthesized as previously described (34).

HEK293T was cultured in DMEM (Corning) with 10% FBS, 1% Penicillin‐Streptomycin (Gibco) and 1% GlutaMAX (Gibco). 8 × 10⁴ cells per well were seeded on 48‐well plates (NEST) and transfected at 70% confluence with 1 µL Lipofectamine 3000 plus 1 µL P3000 according to the manufacturer's protocol. For base editing, 500 ng ABEs plasmid and 500 ng in vitro transcribed sgRNA were used per well. For prime editing, 500 ng PEs plasmid, 125 ng pegRNA, or epegRNA plasmid and with or without 42 ng nicking sgRNA plasmid (PE3 or PE2) were used per well. 72 h after transfection, genomic DNA was isolated. In brief, medium was discarded, and cells were lysed with lysis buffer (10 mM Tris‐HCl, pH 7.5 (Macklin, Shanghai, China), 0.05% SDS (Macklin), 800 units/L proteinase K (NEB) at 37 °C for 1 h, then protease K was inactivated at 80 °C for 20 min.