Sybr green nucleic acid gel stain

In order to analyze the purity of the DNA and resulting DNA/PNA or DNA/PNA/PEG complexes, Polyacrylamide Gel Electrophoresis Electromobility Shift Assays (PAGE-EMSA) was performed. PAGE-EMSA was completed using 5%, 10%, or 4-20% TBE gels in pH 8.3, TBE buffer for 20 minutes at 100 V followed by 0.5-2 hours at 150 V. The gels were stained with Sybr Green nucleic acid gel stain (Life Technologies), and visualized using a Bio-Rad Gel Doc EZ Imager or UV light.

Genomic DNA for mutation determination was extracted from 3 million immortalized T cells using QIAamp^® DNA Mini (Qiagen, Düsseldorf, Germany) and resuspended in H₂O. A total of 100 ng were used for PCR amplification using Easy™ Oligo primers purchased from Sigma Aldrich (Burlington, MA, USA), designed as indicated in Supplementary Table S1, and TAQ PCR MASTER KIT (1,000, Cat. No. 201443) from Qiagen. PCR products were Sanger sequenced in the Genomics Unit of the Complutense University of Madrid (Spain) on a 3730xl DNA Analyzer and studied by Chromas software.
To analyze SMARCAL1 expression, total RNA was isolated using the RNeasy Mini Kit (250, Cat. No. 7410) from Qiagen and resuspended in H₂O. A total of 2 µg was used for reverse transcription using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems™ (Waltham, MA, USA). Different amounts of cDNA were used for PCR amplifications using Easy™ Oligo primers purchased from Sigma Aldrich (Supplementary Table S1) and TAQ PCR MASTER KIT (1,000, Cat. No. 201443) from Qiagen. PCR products were loaded onto a 2% agarose (Tris-acetate-EDTA buffer) gel with a 1/30,000 dilution of SYBR^® Green nucleic acid gel stain from Invitrogen (Waltham, MA, USA) and visualized with the GelDoc Go Gel Imaging System from BIO-RAD (Hercules, CA, USA).

Melted sea-ice samples were fixed in 1% glutaraldehyde (final concentration) and stored at −80°C until analysis. Bacteria were stained with SYBR Green Nucleic Acid Gel Stain (Invitrogen, final dilution 5 × 10⁻⁵) for 10 min in darkness. Cell counting was performed with a FACScalibur flow cytometer (BD Biosciences, Mountain View, USA), using side scattering light (SSC) and green fluorescence (FL1) for detection. Flow rate was determined with an internal standard of 1 μm FluoSpheres (Invitrogen, Eugene, OR, USA). The concentration of FluoSpheres was quantified by triplicated measurement together with BD Trucount absolute counting beads (BD Biosciences, Mountain View, USA). Bacterial abundances were either integrated over area, or corrected to brine volume according to Frankenstein and Garner [40 ].

RNA was isolated from primary rat cortical cell cultures on DIV 2, 7 and 21 using TRIzol Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. RNA was isolated from rat cortical tissue (neocortex) collected on PND 0/1 and 7 from euthanized rat pups. Neocortices from male and female pups of the same litter were pooled, and samples were collected from 3 independent litters (n = 3 biological replicates for each time point). Harvested tissues were placed in RNAlater solution (Ambion/ThermoFisher Scientific) overnight at 4 ^oC and stored at -20 ^oC until further processed. Cortical tissues were thawed on ice, and then homogenized in TRIzol Reagent. RNA was isolated from homogenates according to the manufacturer’s protocol. Following extraction, RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Nucleic acid purity was determined based on the ratio of absorbance at 260 nm to 280 nm (A260/A280 of 1.8–2.2 was considered acceptable), and the absence of genomic DNA contamination was determined by visualization on an agarose gel stained by SYBR Green nucleic acid gel stain (ThermoFisher Scientific). RNA was reverse transcribed at a concentration of 1 μg of RNA per 20 μL reaction, using the Superscript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions.

Chicken DT40 cells were cultured in the presence or absence of 4 mM ddC for 2 h at 37°C. After 2 h, a total of 700 cells were resuspended in 70 μl 0.5% low melting point agarose (Trevigen, 4250-050-02) at a ratio of 1:10 (v/v) and immediately spread on a comet slide (Trevigen, 4250-200-03). Slides were placed flat at 4°C in the dark for 30 minutes. Cells were lysed in a cold lysis solution (Trevigen, 4250-050-01) at 4°C for 1 h. DNA migration was performed in TBE buffer at 1 V cm⁻¹ for 30 min. Slides were washed in milliQ water for 5 min and then fixed with 70% ethanol for 30 min and dried at room temperature. Comets were labeled with SYBR^® Green Nucleic Acid Gel Stain (ThermoFisher) for 30 min at room temperature in the dark. Images were acquired with a confocal fluorescence microscope and analyzed using ImageJ (Open Comet) software. At least 150 comets were scored per sample in each experiment.

To extract RNA, zebrafish were removed from RNAlater and homogenized in 350 μL of RLT buffer in the Qiagen RNeasy kit (Qiagen) with 1 mm glass beads using the Bullet Blender (Next Advance, Troy, NY). Homogenates were transferred to 1.5 mL Eppendorf tubes and spun in a tabletop centrifuge at maximum speed (21000 x g) for 3 min. The supernatant was transferred to 2 mL Eppendorf tubes and RNA was extracted using the Qiagen RNeasy kit and an automated QIAcube robotic workstation (Qiagen) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Nucleic acid purity was determined based on the ratio of absorbance at 260 nm to 280 nm (A260/A280 of 1.8–2.2 was considered acceptable), and the absence of genomic DNA contamination was determined by visualization on an agarose gel stained by SYBR Green nucleic acid gel stain (ThermoFisher Scientific). RNA was reverse transcribed, at a concentration of 1 μg of RNA per 20 μL reaction, using Superscript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions.