GSM6615639: 11- GBP06; Homo sapiens; RNA-Seq
Frozen tissue was embedded in optimal cutting temperature compound and 8 µm sections were frozen sectioned a microtome-cryostat.  Sections were mounted on to a PET membrane steel frame slides (1.4 µm, RNase-free, Leica LMD slides, NCI, Inc. Brooklyn Park, MN) and sectioned slides were stored at -80oC.  Applied 50 µl of RNase Inhibitor (ProtectRNA 500x R7397, Sigma-Aldrich) was added to all staining jars (per 25 ml of the following ethanol and isopropanol jars, not in xylene jar).  Frozen slide was immediately submerged into 95% ethanol for 30 sec. with frequent dipping up and down.  Hematoxylin stain was directly applied to the tissue sections on the slides for 60 sec.  De-stained in 100% isopropanol, 10 dips for 15 sec. Eosin stain was applied directly to tissue sections on the slide for 2 sec and de-stained in 100% isopropanol for 10 dips for 30 sec.  The slides were then de-stained subsequently in 75% ethanol for 5 dips for 15 sec, then 95% ethanol for 10 dips, then 100% ethanol for 10 dips for 10 sec. A final submersion of the slide in anhydrous 100% ethanol for 30 sec.  Then the slide was repeatedly dipped in xylene for 15 sec with a final submersion for 10 min.  Slide was air-dried for 2 min.  Laser capture microscopy immediately followed in which two sections were cut of about 10-30 intestinal epithelial cells along with the villi and collected into the collection cap (Leica LMD 6, Deer Park, IL).  Sectioned cells were lysed with 5µl of lysis buffer the Tecan LCMSeq kit and tubes were stored at -80oC.  RNA library was prepared using the Tecan Ovation SoLo RNA-Seq library preparation kit per manufacturer's instructions. Libraries were quantified using Quant-iT PicoGreen dsDNA assay kit and read on BioTek FLx800 fluorescence microplate reader. Libraries were pooled equal molar and the sample pool was quantified using Biorad ddPCR. Libraries were sequenced using Illumina HiSeq X Ten at Psomagen (Rockville, MD).