RNA-Seq comparing murine intestinal smooth muscle under healthy intestinal epithelium (WT mice) and intestinal smooth muscle under a lawn of intestinal tumors (ApcMin mice)
RNA-Seq comparing murine intestinal smooth muscle under healthy intestinal epithelium (WT mice) and intestinal smooth muscle under a lawn of intestinal tumors (ApcMin mice)
Mice were euthanized by administration of isoflurane followed by cervical dislocation. Following euthanasia, mice were dissected and small intestines removed, rinsed, and stored in fresh ice-cold PBS. The intestines were opened up longitudinally and oriented on a petri dish such that the lumen faced down and serosa/smooth muscle faced up. If the mouse had an ApcMin genotype, the distal 2 cm of the ileum was removed and the 7cm of distal intestine was examined for a robust tumor lawn. If the section of intestine was not sufficiently covered with tumor, the mouse was excluded from the study. The smooth muscle and serosa layers for both WT and ApcMin mice were then separated from the mucosa layer by making a small incision with a round-edged scalpel and gently pulling the muscle away from the mucosal layer. 7cm of distal muscle was dissected and immediately placed in RNAProtect or RNALater for downstream nucleic acid extraction. The tissues were removed from RNAProtect or RNALater and inserted into tissue homogenization tubes (Precellys Soft Tissue Homogenizing kit - P000933-LYSK0-A) with Qiagen RNEasy Plus Mini kit (Qiagen  74134) Lysis Buffer supplemented with beta mercaptoethanol. The tissue was homogenized using a Precellys homogenizer on a program of 2 x 15s, twice. The tubes were centrifuged to remove frothing. Then, the RNA was extracted according to the protocol given in the Qiagen RNEasy Plus Mini kit. 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 a non-directional library. It was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.