Chemidoc mp gel documentation system

Dot blot assay was performed using a published protocol^{64 (link)} with some modifications. Briefly, gDNA was isolated from mouse NSCs or patient fibroblasts using DNeasy Blood and Tissue Kit (Qiagen, 69504) without the RNase treatment. Equal amount of gDNA of each sample (2 µg) was treated with RNase T1 (Thermo Scientific, EN0541), RNase H (NEB, M0297S) or buffer at 37 °C for 1 hr. The samples were spotted on a nitrocellulose membrane, cross-linked using a UV cross linker (GS Gene Linker, Bio-Rad) (120 mJ) and blocked in 5% milk in PBST (1× PBS, 0.1% Tween 20) overnight. The membrane was incubated with rabbit S9.6 antibody (1:1000) diluted in 3% milk, 1% BSA in PBST overnight at 4 °C. Next day, the membrane was washed three times with PBST, incubated with goat anti-rabbit-HRP antibody (Agilent, P0448) diluted in 3% milk, 1% BSA in PBST (1:1000) for 2 h at room temperature, washed three times with PBST and visualized using Clarity Western ECL Substrate on a Bio-Rad ChemiDoc MP Gel Documentation System.

HRP-conjugated secondary antibodies were visualized via chemiluminescence using SignalFire ECL Reagent (Cell Signaling Technology) and the ChemiDoc MP gel documentation system (Bio-Rad).

ITS1-5.8S-ITS2 was amplified from the cell-free DNA lysate using primers ITS1 and ITS4 mentioned elsewhere. The amplification was carried out in a 25 μL final reaction volume containing 50 ng of the genomic DNA as previously described [41 (link)]. The amplified ITS fragment was analyzed by 2.0% (w/v) agarose gel electrophoresis at 80 V in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) buffer to check its intactness and absence of non-specific amplification. The PCR product (4 μL) was digested with 5 U of TaqI (Promega, Madison, USA) in a 10 μL reaction volume at 65°C as per manufacturer’s instructions. The restriction patterns were analyzed by electrophoresis of the 10 μL reaction volume on 2.0% (w/v) agarose gel in parallel with PCR 100 bp Low DNA ladder (Sigma-Aldrich) as molecular size standard. The electrophoresis was run at 80 V for 2 h in 0.5× TBE buffer. The gel was then stained in 0.5 μg/mL ethidium bromide solution for 30 min with rocking at 15 rpm on a platform rocker (Tarsons, Kolkata, India). After destaining for 30 min in sterile deionized water, the gel was photographed using ChemiDoc MP gel documentation system (Bio Rad, Hercules, USA). The restriction fingerprints were analysed for the absence or presence of discriminating fragments using GelCompar II software, version 6.5 (Applied Maths, Sint-Martens-Latem, Belgium).