title: GSM3684899: 16071_0002; Homo sapiens; OTHER
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). The microRNA maturing enzyme Dicer 1 (ribonuclease III, Gene ID 23405 on Chromosome 14) was defined as the target gene. The online tool Illumina Design Studio (http://designstudio.illumina.com/) was used to design an amplicon-based enrichment panel (TruSeq Custom Amplicon Low Input) that targets the DICER1 gene. The design strategy was based on human reference genome hg19 including the coding sequence of DICER1 with a targeted amplicon size of 175 bp (13). Library preparation was performed with the TruSeq Custom Amplicon Low Input Library Kit (Illumina, San Diego, USA) using a dual pool approach according to the manufacturer´s protocol (1). Hybridization of oligos to genomic DNA was performed in a 96-well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers, DNA templates were amplified by PCR and elongated with barcodes and sequencing adapters. In addition to the 16 gDNA samples, resulting in 32 DNA-oligo-pool samples, a control DNA (2800M) and a non-template control (NTC) were processed in parallel in order to control the complete library preparation process. Identification of each sample was ensured by an unique index sequence combination allowing identification of each sample. The DNA 1000 and High Sensitivity LabChip Kit (Agilent Technologies, Santa Clara, USA) were used on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) to analyze the quality and fragment distribution of the generated libraries. The expected PCR product size including adapter sequences was ~ 280 bp for the targeted amplicon size of 175 bp. bidirectional sequencing was performed, starting first at the end of the sense strand (read 1) and subsequently at the end of the complementary strand (read 2). Both reads had a length of 150 bases, finally producing 300 bases of sequence information in 2 x 150 bp paired-end (PE) reads.
identifiers.namespace: GEO
identifiers.id: GSM3684899