title: GSM3684931: 16071_0010_RNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Genomic DNA (gDNA) and total RNA were isolated in parallel from the same sample using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. An on-column DNase digestion step was included in the RNA isolation protocol. The final volume of the eluate was 50 μl for gDNA and 30 μl for total RNA. The total RNA samples were isolated for RNA and miRNA sequencing analysis. An aliquot of each total RNA sample was used to determine RNA concentration and purity on the NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). All samples were analyzed on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) using RNA 6000 Nano LabChip Kits (Agilent Technologies, Santa Clara, USA) and RNA integrity number (RIN) was determined.   An aliquot of each gDNA sample was used to determine purity and approximate DNA concentration on the NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). The TruSeq Custom Amplicon Low Input (TSCA) library preparation protocol was followed. The gDNA samples were additionally quantified using the highly sensitive fluorescent dye-based Qubit® dsDNA HS Assay Kit (ThermoFisherScientific, Karlsruhe, Germany). For mRNA sequencing analysis, one RNA sequencing library was prepared from five total RNA samples (sBCC samples 10-14) from fresh frozen tissue with the Illumina TruSeq® Stranded mRNA HT technology. Library preparation for the total RNA samples isolated from fresh frozen tissue was performed with the TruSeq® Stranded mRNA HT technology (Illumina, San Diego, USA), according to the manufacturer´s protocol. For small RNA sequencing, one small RNA sequencing library was prepared from five total RNA samples derived from FFPE tissue samples (sBCC samples 10-14) using the NEBNext® Small RNA Library Prep Kit for Illumina. The kit targets microRNAs and other small RNAs that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes. Sequencing of all libraries was performed at a final concentration of 1.8 pM and with a 1% PhiX v3 control library spike-in on the NextSeq500 sequencing system (Illumina, San Diego, USA). For cluster generation and sequencing of all samples, three high output (HO) single-end 75 cycles (1x75bp SE) runs were performed for the total RNA and mRNA  libraries. Sequencing was operated under the control of the NextSeq Control Software V2.1.0 (Illumina, San Diego, USA). The resulting mRNA and totalRNA reads were quality controlled and mapped against the human reference genome. In sum, the achieved quality values of the NextSeq500 sequencing runs met the manufacturers given specifications (Illumina, San Diego, USA). All data could be used for in depth data analysis.
identifiers.namespace: GEO
identifiers.id: GSM3684931