GSM9700378: HFD, PCCA Vill, replicate 3; Mus musculus; RNA-Seq
Colon sections were dissected from mice and flushed with chilled PBS before transfer to 2ml eppendorf tubes and flash-freezing in liquid nitrogen. Samples were stored at -80 degrees Celsius until RNA extraction. Tissues were bead homogenized in Trizol reagent (11596018, Invitrogen) and processed according to manufacturer protocol. Total RNA was extracted by isopropanol precipitation followed by ethanol washes, drying of the pellet, and resuspension in RNAse-free water. RNA quality was assessed by Nanodrop before packaging and submission for RNAseq analysis at Novogene Corporation Inc. RNA was submitted to Novogene Corporation Inc for library construction and sequencing according to their protocol: "Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dTTP for non strand specific library or dUTP for strand specific library. For the non strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification."