title: GSM8894801: H2030 cells, biol rep 1; Homo sapiens; RNA-Seq
library_name: GSM8894801
library_construction_protocol: The high brain metastasis derivative of H2030 cell line (H2030-Lbrm) were isolated from mouse metastatic brains derived from an experimental brain metastasis model using H2030 cells and further cultured in vitro. The cells were maintained in RPMI-1640 medium (C22400500BT, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (A5669701, Gibco) at 37°C within a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% CO2. Cells in the logarithmic growth phase were selected for assays and confirmed as mycoplasma-free. Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) Shanghai Personal Biotechnology Cp. Ltd.