title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of mouse EL4 cells overexpressing Israa gene product in comparison with Mock-transfected control cells
design: RNA-seq of mouse EL4 cells overexpressing Israa gene product in comparison with Mock-transfected control cells
library_construction_protocol: EL4 (Mus musculus lymphoma, ATCC ID: TIB 39) mouse T-cell lymphoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% horse serum (Gibco, Gaithersburg, MD, USA), 0.1 mg/ml streptomycin and 100 U/ml penicillin at 37°C with 5% CO2. Cells were grown to reach a density of 2.5x106 cells/ml. A total of 2x107 cells were mixed with 10 μg of either p-SELECT-zeo-ISRAA expressing N-terminal 6His-tagged ISRAA or p-SELECT-zeo-eGFP. The cell suspension (0.4 ml) was subjected to electroporation using a BTX Electro Cell Manipulator with the following parameters; 140 V, 720 Ohms, and 3175 μF, across a BTX cuvette with a 0.2 cm electrode gap. Pulse lengths varied between 25 and 28 ms. Electroporated cells were incubated at room temperature for 15 minutes, then transferred to complete medium and returned to the cell culture incubator. Zeocin was subsequently added for selection at concentrations ranging from 100 to 200 μg/ml. The electroporation efficiency was assessed by fluorescence microscopy with the p-SELECT-zeo-eGFP electroporated live cells.  Ten days after transfection, 2x105 cells were harvested and assayed for recombinant ISRAA expression by Western blotting and total RNA extraction. RNA extraction using Rneasy Mini Kit (QIAGEN) For RNA-seq analysis, 5 μg of total RNA was isolated using an RNeasy Mini kit (QIAGEN) according to the manufacturer's recommendations. RNA purification, reverse transcription, library construction and sequencing were performed according to the manufacturer's instructions (Illumina, San Diego, CA). Two RNA-seq transcriptome libraries (ISRAA-expressing EL4 cells and non-electroporated control EL4 cells) were prepared using a modified Illumina TruSeq RNA Sample Preparation protocol. The first step involves the removal of ribosomal RNA (rRNA) using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. The Ribo-Zero Gold kit (Illumina, San Diego, CA) depletes samples of both cytoplasmic and mitochondrial rRNA. Following purification, the mRNA is fragmented using divalent cations under elevated temperature. Taking these short fragments as templates, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina, USA). Then, the synthesized cDNA was subjected to end-repair, phosphorylation, 'A' base addition and ligation of the adapters according to Illumina's library construction protocol. Libraries were size- selected for 200–300 bp cDNA target fragments on 2% Low-Range Ultra Agarose followed by PCR amplification using Phusion DNA polymerase (New England Biolabs, Boston, MA).
attributes.tag: Experimental Factor: phenotype
attributes.value: overexpression of ISRAA