title: Environmental toxins and colon carcinogenesis.
design: RNA sequencing libraries were prepared with Illumina-compatible NEBNext  Ultra  Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 200ng of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis in the presence of Actinomycin D (Gibco, life  technologies, CA,  USA) followed  by  second  strand synthesis. The  double  stranded  cDNA  was purifiedusing HighPrep magnetic beads (Magbio Genomics Inc, USA). Purified cDNA was end-repaired, adenylated  and  ligated  to  Illumina  multiplex  barcode  adapters  as  per  NEBNext   Ultra  Directional RNA Library Prep Kit protocol.  Adapter  ligated  cDNA  was  purified  using HighPrep beads  and  was  subjected  to  14cycles  of Indexing-PCR (37   C for 15mins followed by denaturation at 98  C for 30 sec, cycling (98  C for 10sec, 65  C for 75sec) and 65  C for 5mins) to  enrich  the  adapter-ligated  fragments. Final  PCR  product (sequencing  library)  was  purified  with  HighPrep  beads,  followed  by  library  quality  control  check. Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA,  USA)  and  its  fragment  size  distribution  wasanalyzed  on Agilent 2200 Tapestation