title: GSM9478813: EpiVaginal tissue, 150 nM testosterone, T1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using the RNAqueous Micro Kit (Invitrogen).  Modifications to the manufacturer's protocol for the MatTek tissues were as follows.  The microporous membrane from the cell culture inserts was cut away using sharp forceps.  It was added to lysis buffer and homogenized using a pellet pestle mixer tip.  RNA extraction was then performed according to the manufacturer's instructions.  Total RNA samples are quantified using Nanodrop and qualified by agarose gel electrophoresis. Samples were quantified on a NanoDrop ND-1000 instrument. Agarose electrophoresis was used to check the integrality of total RNA samples. 1 - 2 ug total RNA of each sample was taken for RNA-seq library preparation. Briefly, mRNA was isolated from total RNA with NEBNext® Poly(A) mRNA Magnetic Isolation Module. Alternatively, rRNA is removed from the total RNA with a RiboZero Magnetic Gold Kit; The enriched mRNA or rRNA depleted RNA was used for RNA-seq library preparation using KAPA Stranded RNA-Seq Library Prep Kit (Roche). The library preparation procedure included: 1) Fragmentation of the RNA molecules; 2) Reverse transcription to synthesis first strand cDNA; 3) Second strand cDNA synthesis incorporating dUTP; 4) End-repair and A-tailing of the double stranded cDNA; 5) Illumina compatible adapter ligation; 6) PCR amplification and purification for the final RNA-seq library. The completed libraries were qualified on Agilent 2100 Bioanalyzer for concentration, fragment size distribution between 400-600 bp, and adapter dimer contamination. The amount was determined by absolute quantification qPCR method. The barcoded libraries were mixed in equal amounts and used for sequencing on the instrument.