Nanodrop 8000

Fecal samples were collected by the subjects in a sterile container (HiMedia, India) and stored in -80°C freezer until DNA extraction. Genomic DNA was extracted from stool samples using QIAamp DNA stool minikit (Qiagen, Hilden, North Rhine-Westphalia, Germany) following manufacturer’s instructions with a few modifications. The fecal sample in the sterile container was mixed manually using a sterile spatula (HiMedia, India) until it formed a homogeneous mixture. Then 300 mg of sample was transferred into a 2 ml centrifuge tube and extraction was carried out according to the protocol. The extraction was performed in triplicates for each sample. At the final step, DNA was eluted with 100 µl of AE buffer provided by Qiagen. Then, equal volume of DNA was taken from each replicate and pooled together for PCR amplification and sequencing. Quality of genomic DNA was checked on 0.8% agarose gel and quantification was performed using Nanodrop 8000 (GE Healthcare, India) and Qubit 2.0 fluorometer with the help of Qubit dsDNA HS Assay kit (Life Technologies, India).

The transcriptomic analysis has been carried out using RT² Profiler PCR Array, a commercially available 96-well plate using real-time PCR (RT-qPCR) approach. In brief, HepG2 cells were seeded in 6-wells plate and treated with 100 µM of TEHP for 72 h. Total RNA was isolated by using RNeasy Mini Kit (Qiagen, Germantown, MD, USA). Purity of RNA was measured on Nanodrop 8000 and cDNA synthesis was carried out using ready-to-go PCR beads (GE Healthcare, Chalfont Saint Giles, UK). Transcriptomic changes of 84 genes belonging to human cancer pathway was analyzed by RT² Profiler™ PCR Array (PAHS-033Z, SA Biosciences Corporation, Allentown, PA, USA) on Roche^® LightCycler^® 480 (Roche Diagnostics, Rotkreuz, Switzerland). Expressional changes in TEHP-treated cells were normalized with five housekeeping genes and the results are represented heat map, scatter plot, and fold change [29 (link)].