Nucleospin tissue

Genomic DNA was extracted from 200 μL of whole blood and urine using a Nucleospin Tissue (Macherey-Nagel, Duren, Germany) on QIAcube instrument (Qiagen, Hilden, Germany) with final elution volume of 100 μL, according to the manufacturer’s instructions.

Genomic DNA was extracted using DNA isolation kit (NucleoSpin Tissue, Macherey-Nagel GmbH &Co., Düren, Germany). PCR reaction was performed using a Master Mix (Promega, WI, USA) following the manufacturer instructions and analysis using GFP-specific primers, forward 5′- TGAGCAAGGGCGAGGAGC -3′ and reverse 5′-GGAATTCCATATTTGTACAGCTCGTCCATGCCG-3′, GAPDH-specific primers, forward 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ and reverse 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (Sigma-Aldrich). Cycling was done at 95° C for 5 min, followed by 35 cycles of 95 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and a final elongation reaction of 72 °C for 10 min. DNA products were separated in 1% agarose gel (Lonza, Rockland, ME, USA), stained with SYBER (ThermoFisher Scientific, Pittsburg, CA, USA) and visualized and pictures under UV illumination using G-Box Syngene system (Synoptics Group, Frederick, MD, USA).

DNA was extracted using a commercial kit (Genomic DNA from tissue, NucleoSpin^®Tissue from Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. For each extraction lot, a negative extraction control was prepared by using “Buffer T1” instead of the sample. Extracts were stored at -20°C until further analysis was performed. Extracted DNA was measured on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to determine DNA quantity and quality (260/280 value).

Total cellular DNA was isolated from cultured KGN cells using NucleoSpin^® Tissue (MACHEREY‐NAGEL GmbH &Co) according to the manufacturer's instructions. Copy number of mitochondrial DNA (mtDNA) was estimated by real‐time PCR analysis using the mitochondrial genes NADH dehydrogenase subunit 1 (ND1) and ND5. ND1 and ND5 levels were normalized to half the level of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) since each cell contains two copies of genomic DNA compared to a single copy of DNA per chromosome. Each sample was run in triplicate, and real‐time PCR analysis was performed as described above. Primer sequences are reported in Table 1.

After pressurization or chemical treatment, cells were washed twice with ice-cold PBS and lysed in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 0.15 mM Spermine and 0.5 mM Spermidine) for 10 min on ice. Nuclei were washed with digestion buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM Spermine amd 0.5 mM Spermidine) and resuspended in 1 mM CaCl₂-containing digestion buffer. For chromatin digestion, micrococcal nuclease (3 units, New England Biolabs, Inc.) was added to nuclei suspension (100,000 cells in 100 μl) and incubated for 15 min at 37°C. Chromatin digestion was stopped by adding 25 μl of stopping buffer (100 mM EDTA, 10 mM EGTA), followed by protein digestion in 1 mg/ml Proteinase K and 1% SDS (v/v) for overnight at 37°C. Digested genomic DNAs were purified with NucleoSpin Tissue (Macherey-Nagel GmbH & Co. KG) and quantified with a Qubit assay (Thermo Fisher Scientific). Equal amounts of genomic DNAs (25 ng) for each sample was analyzed Agilent 2200 TapeStation with Genomic DNA Screen Tape (Agilent Technologies).

Genomic DNA was isolated from frozen cell pellets using the NucleoSpin^® Tissue (MACHEREY-NAGEL) according to manufacturer instructions. Fifty ng of genomic DNA in total 50 μl reaction mixture were digested with methylation-sensitive HpaII at 37 °C for 12 h, and 2 μl of reaction mixture were then subjected to real-time PCR using primer set (shown in S2 Table) to amplify a fragment including CpG sites in Myod1 promoter region with iTaq^® SYBR™ Green Supermix with ROX (BIO-RAD, Hercules, CA, USA). PCR reactions were carried out using 7500 Real-Time PCR System (Applied Biosystems) under the following standard conditions: Three independent measurements were averaged and relative gene expression levels were calculated as a ratio to sample DNA at Set0.

Genomic DNA was isolated using NucleoSpin^® Tissue (catalog# 740952, Macherey-Nagel, Allentown, PA, USA) from cells treated with LV_crMYOC, AAV2_crMYOC, LV_Null and AAV2_Null. Untreated cells were used as experimental control. MYOC, which is a target of selected gRNA was amplified by PCR. PCR product was denatured and reannealed using the Alt-R^™ Genome Editing Detection Kit protocol (catalog# 1075932, Integrated DNA Technologies, Coralville, Iowa, USA). This generated mismatched heteroduplex DNA products containing strands with CRISPR/Cas9-induced indel reannealed to wild-type strands or different indel. The heteroduplexes were subsequently detected using T7 endonucleases (T7E1), that cleaved the mismatched DNA. The resulting cleaved products were analyzed by gel electrophoresis.