title: GSM5421409: PVR S6; Homo sapiens; RNA-Seq
library_construction_protocol: RNA extraction, library preparation and RNA sequencing were performed of 28 samples in total at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de). In short, membranes for RNA extraction were directly transferred into 50 µl RNAlaterTM (ThermoFisher). After the shipping, RNAlaterTM was replaced by RLT Plus buffer and the obtained tissue homogenized by vortexing for 30 seconds. Using gDNA Eliminator spin columns eliminated Genomic DNA contamination. After adding ethanol the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Lastly, total RNA was eluted in 12 μl of nuclease-free water. 750 pg total RNA was converted to first-stranded cDNA using the SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc.) 12 cycles of LD PCR were performed to amplify double-stranded cDNA followed by purification via magnetic bead clean-up. The Library was prepared according to Illumina Nextera XT Sample Preparation Guide (Illumina, Inc.). 150 pg of cDNA were tagged and fragmented via Nextera XT transposome. After adding partial adapters a limited-cycle PCR program was used to amplification resulting in multiplexed sequencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc.). The Illumina TruSe SR Cluster Kit v3 was used for cluster generation on the cBot with pooled equimolar amounts of each library. The products were sequenced on a HiSeq1000 instrument with TruSeq SBS Kit v3 according to the Illumina HiSeq 1000 System User Guide. Illumina image analysis and base calling were recorded and converted to Fastq files via the CASAVA1.8.2/ bcl2fastq v2.18 software.