GSM1666034: Control 0, ZH1, or B12; Homo sapiens; RNA-Seq
Biopsies were collected on site, washed briefly with HBSS and immediately oriented in OCT compound (Sakura, Torrence, CA). Samples were frozen by immersion in pre-cooled isopentane in an acetone bath encased with dry ice. Laser capture microdissection was performed to isolate the vaginal epithelium for RNA analysis. Sections were cut at 8 um on PEN slides under RNAse free conditions. Slides were incubated for 30 seconds in EtOH, 30 sec in RNAse free dH20, and stained with cresyl violet for 45 sed. Slides were then dehydrated in progressively higher EtOH solutions and xylene. LCM was performed within 15 minutes of staining on an Arcturus Veritas 704 microdissection system with HS caps. Vaginal epithelial cells were precisely removed without an lamina propria cells. A Picopur RNA Isolation kit was used to isolate RNA from the tissue with an RNAse free DNase Set used to remove contaminating DNA. mRNA-sequencing was performed on the Illumina HiSeq2000, providing up to 300Gb of sequence information per flow cell. Total RNA was assessed using the Agilent 2100 Bioanalyzer followed by 2 rounds of poly A+ selection and conversion to cDNA. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation prior to cluster generation. Clusters were generated to yield approximately 725K-825K clusters/mm2. Paired end 2 x 50bp sequencing runs were performed to align the cDNA sequences to the reference genome.
GEO Accession
GSM1666034