GSM9409884: Vulvar Swab 3; Homo sapiens; RNA-Seq
We collected vulvar specimens  by a gentle swab of the vestibule area in a 1.5ml tube with RNA/DNA shield reagent to preserve nucleic acids. RNA extraction was performed by adding 1 ml of QIazol/Trizol Lysis Reagent (79306 Qiagen). After incubation at RT for 3 minutes, 200ul of Chloroform were added to samples which were vortexed for 15 seconds and incubated at room temperature for 3 minutes. The samples were then centrifuged at max speed for 15 minutes at 4°C, and the obtained aqueous phase was transferred into a new tube and precipitated with 500ul of Isopropanol and 1ul of Glycogen at room temperature. After 10 minutes samples were centrifuged at maximum speed for 15 minutes at 4°C. The pellet was washed with  500ul of 70% EtOH, resuspended in 30ul of DNAse/RNAse-free water. For DNA, the Vulvar swabs were  soaked  in 1 ml of NaCl 0.9% for 5 minutes, while vortexing. After centrifugation at 13000 rpm for 10 minutes at room temperature, the supernatant was discarded and the pellet was resuspended in 80ul of TrisHCl. Subsequently 20ul of lysozyme (10mg/ml) were added and samples were kept at 37°C for 30 minutes to allow cell wall degradation. After incubation, we performed bead beating using TissueLyser II at 30Hz for 2 minutes with 1mm metallic beads. Next, to remove proteins, we added 1ul of proteinase K (20 ug/ul) and we incubated samples at 56°C for 30 minutes. To extract DNA, we added 500 ul of Phenol-Chloro-Isoamyl-Alcohol and 500ul of NaCl 0,9%, incubated them at room temperature for 10 minutes and then centrifuge at 11000 rpm for 5 minutes. After phase separation, we added to the aqueous phase 1ul of glycogen, 30 ul of SodiumAcetate 3M and 800ul of EtOH 100% and we precipitated at -20°C overnight.  The next day, to purify the DNA, samples were centrifuged at 13000 rpm at 4°C for 30 minutes, the supernatant was removed and the samples were washed with 500 ul of EtOH 70%. After centrifugation at 13000 rpm at 4°C for 5 minutes, the supernatant was discarded and the pellet was resuspended in 30 ul of RNAse-free water. RNA-Seq libraries were prepared exclusively using the Illumina Stranded mRNA prep kit. For DNA-seq libraries, 100 ng of DNA were sonicated at the Pico Bioruptor (Biosense srl) with 3 cycles of sonication (20 seconds ON and 30 seconds OFF). Libraries were prepared using Watchmaker DNA Library prep kit, following the manufacturer's instructions