title: 0.15 M SUCROSE DIET
design: RNA sequencing libraries were prepared with Illumina-compatible SureSelect Strand-Specific RNA Library Prep kit (Agilent, Santa Clara, CA, U.S.A.) at the Genotypic Technology Pvt. Ltd., Bangalore, India. Briefly, mRNA enrichment was performed from 1  g of Qubit quantified total RNA. The enriched mRNA was fragmented for 4 minutes at 94oC in the presence of divalent cations. First strand cDNA was synthesized in the presence of Actinomycin D (Gibco, life technologies, Carlsbad, CA, U.S.A.) and subsequently the single stranded cDNA was purified using HighPrep magnetic beads (Magbio Genomics Inc, USA). Purified single stranded cDNA was used for second strand synthesis followed by end repair using Second Strand Synthesis + End Repair mix. The end repaired cDNA was purified using HighPrep magnetic beads. Adapters were ligated to the cDNA molecules after 3  Adenylation. The adapter-ligated DNA was purified with HighPrep beads and then amplified for 10 cycles of PCR using Illumina-compatible indexing primers provided in the SureSelect Strand-Specific RNA Library Prep kit. The final PCR product (sequencing library) was purified with HighPrep beads, followed by library quality control check.  The sequencing library was initially quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent TapeStation. Finally, the sequencing library was accurately quantified by quantitative PCR using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). The qPCR quantified libraries were pooled in equimolar amounts to create a final multiplexed library pool for sequencing on an Illumina sequencer for 75 bp paired-end chemistry.