GSM5289844: Rat_6_con_distal; Rattus norvegicus; RNA-Seq
After 10 days on the different diets rats were euthanized by cervical dislocation, their abdomens opened and the colons were dissected out and flushed clean off faeces with cold PBS. Colons were divided into thirds and the middle section discarded. The proximal segment (initial 1/3) and the distal segment (last 1/3) were inverted, tied together at both ends and filled with Ca2+ free Ringer buffer with EDTA to form small sacs. The colon sacs were put in separate 50 mL tubes with warm Ca2+ free Ringer buffer with EDTA and incubated at 37°C for 20 min. The tubes were shaken vigorously to release colonic epithelial cells (CECs) from the colon sacs. Colon sacs were then removed and the CEC suspensions were divided into two tubes and centrifuged at 4000 g for 3 min to pellet the cells. Supernatants were discarded and pellets were resuspended and homogenized in either 1 mL TriReagent™ (Ambion®) for RNA-analysis or in dissection buffer for protein analysis. TriReagent samples were sonicated and RNA was extracted using the RiboPure™ RNA Purification kit (Ambion®) according to the manufacturer's instructions. RNA yield was estimated based on A260 measurements by spectrophotometry using a NanoPhotometer® (Implen) with Elution Buffer from the RiboPure™ kit as blank. Preparation of strand-specific cDNA libraries, RNA selection using polyA purification and single channel RNA sequencing was performed by Eurofins Genomics (Germany) using HiSeq SBS Kit v4 on a HiSeq 2500 (Illumina).