Buffer r

Human MDMs were electroporated with PBS, mouse anti-GFP (9F9.F9; Abcam) or mouse anti-NLRP3 (Cryo-2; Adipogen) IgG antibodies. All antibodies used for electroporation were passed through Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace buffer with PBS. All antibodies were diluted to 0.6 mg/mL in PBS prior to electroporation. Antibody electroporation was performed using the Neon Transfection System (Thermo Fisher Scientific). MDMs were washed with PBS and resuspended in Buffer R (Thermo Fisher Scientific) at a concentration of 1.4 × 10⁸ cells/mL. For each electroporation reaction 1.4 × 10⁶ cells (10 μl) were mixed with 2 μl of antibody or PBS. The mixture was taken up into a 10 μl Neon® Pipette Tip (Thermo Fisher Scientific) and electroporated using the following settings: 1400V, 20 ms, 2 pulses. Electroporated cells were transferred to growth medium without antibiotics.

Cells were grown to 80%–90% confluence, and then were harvested using Trypsin/EDTA Solution (Thermo Fisher Scientific, Carlsbad, CA, USA), re-plated, and cultured for 24 h. On the day of transfection, cells were harvested, washed twice in DPBS (Thermo Fisher Scientific, Carlsbad, CA, USA), and resuspended in Buffer R (Thermo Fisher Scientific, Carlsbad, CA, USA). Electroporation was performed using the Neon Transfection System (Thermo Fisher Scientific, Carlsbad, CA, USA) with a time constant protocol at 1400 V for 30 ms per 1 million of HDF-A cells and 1700 V for 20 ms per 0.75 million of HDF-N cells per transfection. Each cell line was transfected with 30 nM of one of six small interfering RNAs (siRNAs), targeting transcripts of CTGF, TGFBR2, TGFB1, TGFB3, DNMT1, and DNMT3A (Santa Cruz Biotechnology, Dallas, TX, USA). A randomized siRNA (Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene product was used as an internal control. Detailed information about the applied siRNAs is shown in Supplementary Materials Table S1.

Initially, 50 μg pSG483 was nicked by incubation with 10 units Nb.Bpu10I (ThermoFisher) in 1× Buffer R (ThermoFisher) for 4 h at 37°C. The enzyme was deactivated by a further incubation step at 80°C for 20 min. The reaction was allowed to cool to room temperature before being supplemented with ATP to a final concentration of 1 mM. 10 μl T4 DNA ligase was added and the reaction was left at room temperature for 16 h. After ligation, ethanol precipitation was performed to remove proteins. An equal volume of UltraPure™ phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol) (ThermoFisher) was added to the reaction mixture before vortexing briefly. The sample was centrifuged at 16 000 × g for 2 min and the resulting aqueous layer was removed and carried forward. An equal volume of chloroform (ThermoFisher) was added to the aqueous layer before centrifugation at 16 000 × g for 2 min. The resulting aqueous layer was carried forward and 1/10 volume 3 M sodium acetate pH 5.2 was added. Then, 2 volumes of 100% ethanol were added, briefly mixed by pipetting, and stored at -80°C for 30 min. The sample was centrifuged at 16 000 × g and 4°C for 20 min. The ethanol was removed, and the DNA pellet dried at room temperature. The DNA pellet was resuspended in room temperature dH₂O to approximately 300 ng/μl.

To obtain Cas9-sgRNA RNPs, 1 μg of Cas9 protein (PNA bio) was incubated with 1 μg of synthetic sgRNA (Synthego) for 20 min at room temperature, then 1 μg of PCR-amplified HDR DNA was added to the RNP mixture. 2.50 × 10⁵ OCI-AML3 cells were electroporated in buffer R (Thermo Fisher) using the Neon Transfection System. The following electroporation conditions were used for OCI-AML3 cells: 1,400 V, 10 ms, three pulses.

For nicking, 10 μg pSG483 was incubated with 10 units of Nb.Bpu10I (ThermoFisher) in 1 x Buffer R (ThermoFisher) for 1 hr at 37°C. The enzyme was deactivated by a further incubation step at 80°C for 20 min. For linearisation, 10 μg pSG483 was incubated with 10 units of BamHI-HF® (NEB) in 1 x CutSmart buffer (NEB) for 1 h at 37°C. The enzyme was deactivated after incubation by a further incubation step at 65°C for 10 min. Conversion of supercoiled pSG483 into appropriate products was assessed by agarose gel electrophoresis. Both nicked and linear form pSG483 were subsequently stored at −20°C.

To linearize the different plasmids, an XhoI restriction site downstream of the poly-A tail was used. Plasmids (10 µg) were linearized with 0.5 U/µL XhoI (Thermo Fisher, Waltham, MA, USA) in buffer R (Thermo Fisher, Waltham, MA, USA) for 2 h. The linearized DNA was purified with a 25:24:1 (v/v) mixture of phenol, chloroform, and isoamyl alcohol (Sigma-Aldrich, St. Louis, MO, USA), followed by a chloroform wash, ethanol sodium acetate precipitation, and additional washing with ethanol (75%). For in vitro RNA transcription, the mMESSAGE mMACHINE™ T7 transcription kit (Thermo Fisher, Waltham, MA, USA) was used according to the manufacturer’s instructions, with 1 µg linearized DNA and 1 µL GTP (30 mM). The in vitro transcribed RNA was purified with Microspin™ S-400 HR columns (Cytiva, Marlborough, MA, USA) and stored at −80 °C until further use.