title: NCH644 No HN 2
organism: Homo sapiens
description: Protocols: Glioblastoma cell cultures were derived from human glioblastoma biopsies and were maintained under stem cell cultivation conditions in either NeuroCult Basal Medium (Cat. 05700) with Proliferation Supplement (Cat. 05701 both, STEMCELL Technologies) or DMEM-F12 (Cat. 11320-074) with 1× B27 (Cat. 17504-044), supplemented with 5% penicillin-streptomycin (Cat. 151140-122; all ThermoFisher Scientific, Waltham, MA, USA), 10 ng/ml epidermal growth factor (EGF; Cat. 236-EG; Biotechne; Minneapolis, MN, USA) and 10 ng/ml fibroblast growth factor (FGF; Cat. 100-18B PeproTech, Hamburg, Germany). All cells were maintained at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. HN1 samples where treated with 200nM humanin at day 0 and day 2 and were collected at day 3. Cntrls where vehicle treated only. HN48h samples were treated with 40nM Humanin at day 0 and collected after 48h. No HN samples were vehicle treated and collected at 48h. Total RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions including on-column DNase digestion. Total RNA was eluted in 30 μl RNase-free water. An aliquot of each total RNA sample was used to determine RNA concentration and purity on the NanoDrop ND-1000 spectral photometer (Peqlab). All samples were analyzed on the 2100 Bioanalyzer (Agilent Technologies) using RNA 6000 Nano and Pico LabChip Kits (Agilent Technologies). Library preparation was performed with the RNA samples using the TruSeq® Stranded mRNA HT technology, according to the manufacturer´s protocol (Illumina Inc. TruSeq® Stranded mRNA Sample Preparation Guide - Catalog # RS-122-9004DOC - Part # 15031047 Rev. E. 2013). The protocol started with a RNA fragmentation step using divalent cations. Samples were then introduced into a reverse transcription to generate first strand cDNA. In the second strand cDNA synthesis dUTP is incorporated instead of dTTP, to avoid amplification by the DNA polymerase in the subsequent PCR and thus guarantee strandedness. 3'-ends were adenylated and sequencing adapters were ligated. Those comprise sequencing primer- and flow cell-binding sites, as well as indices for multiplexed sequencing of pooled libraries. Adapter-ligated fragments were amplified during a limited-cycle PCR reaction. After the limited cycle PCR and at the end of the library preparation all samples were quality controlled using the DNA 1000 LabChip kits on the 2100 Bioanalyzer (Agilent Technologies). Furthermore, all libraries were quantified using the highly sensitive fluorescent dye-based Qubit® ds DNA HS Assay Kit (Invitrogen). Afterwards, all single libraries were pooled into a final sequencing library with an equal DNA amount per sample. The final sequencing library, generated by pooling was quantified again using the highly sensitive fluorescent dye-based Qubit® ds DNA HS Assay Kit (Invitrogen). After quantification, the final library was diluted to 2 nM, followed by denaturation with NaOH. This ensures the presence of single stranded DNA fragments for cluster generation. The final sequencing library consists of single stranded fragments with sequencing adapters, sequencing primer binding sites and indices.