distal colons were preserved snap frozen in RNAlater, homogenized in 1 mL of Trizol Reagent (Invitrogen cat. #15596-026) with a 5 mm stainless steel bead (Qiagen cat. #69989) in RNase-free tubes (Ambion) on a tissue homogenizer (Tissue Lyser II) for 3 runs X 2 minutes at frequency 25.0 for 5 minutes at room temperature to permit complete dissociation. 200 μL of chloroform (EM Science cat. # CX1055-6) per mL of Trizol Reagent was added, capped securely and shaken vigorously by hand for 15 seconds. Tubes were incubated at room temperature for 15 minutes and then centrifuged at 10,000 x g for 10 min. at 4 degrees C. The top aqueous phase was harvested after centrifugation and transferred to 96 well deep well plates and stored at -80 degrees C until RNA isolation. RNA was isolated with automation on the Biomek using Agencourt RNAdvance Tissue Kit (Beckman Coulter #14073200). Yield and quality of RNA were determined using Nanodrop and RNA Integrity Number (RIN) values were determined using Tapestation (Agilent)
Label
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Label protocol
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Hybridization protocol
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Scan protocol
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Data processing
All samples were normalized to the same concentration and a total of 100 ng of RNA per sample were used for hybridization and run on the XT_PGX_MmV2_Inflammation_CSO 248 gene panel (NanoString Item No. 115000082)
