With the aid of a dissecting microscope the large intestine was manually dissected from embryos submerged in phosphate buffered saline (PBS). The isolation of epithelium is as described by Nagy et al (2003). The large intestines were then transferred to a Petri dish containing 200 ul of 1% trypsin in Hanks solution. The trypsin was allowed to act on the tissue for 20 minutes at room temperature. The digestion process was stopped with the addition of ~1 ml of 20% fetal bovine serum (FBS) in Hanks solution plus a small amount of DNAse powder. The caecum was removed and not included in the tissue collection. Epithelial tissue was teased away from mesenchyme tissue with either tungsten needles or fine forceps. Epithelial tissue was then collected into Trizol. Epithelium from embryos of several litters was collected to obtain enough RNA for SAGE library construction.
SM155 and SM156 were constructed using the same RNA source, with 2500 ng and 500 ng DNase I treated RNA respectively, ditags were amplified
for 27 cycles in both libraries, tags were extracted and correlated.