GSM5411918: GF_RNA_rep2; Mus musculus; RNA-Seq
To prepare epithelial cell pellets, colons were incubated in PBS containing 30 mM EDTA and 1.5 mM DTT for 20 minutes at 4°C with occasional inversion.  Tissue was subsequently transferred to PBS containing 30 mM EDTA, incubated for 10 mins at 37°C, and shaken vigorously for 1-2 minutes until the mesenchyme separated from the epithelium.  The mesenchyme was removed and epithelial cells were pelleted at 1500 rpm for 5 minutes at 4°C and washed in PBS.  Cell pellets were further incubated with dispase (Corning, 0.3 U/ml) in PBS (100 l dispase per 50 ml PBS) at 37°C for 10 mins and then quenched with 1/10 volume of FBS containing DNase followed by washing in PBS.  Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until use.    Total RNA was extracted from epithelial cell pellets using the RNeasy kit (Qiagen) with QIAshredder columns. The NEBNext Ultra or Ultra II Directional RNA Library Prep Kit for Illumina with the mRNA magnetic isolation module (New England Biolabs) was used to prepare sequencing libraries from 1 µg of total RNA.