GSE122633
Over expression of Receptor Activity Modifying Protein 2 in HEK293T cells
HEK293T cells were transfected with pcDNA3.1 vector containing codon optimized sequence for (Receptor Activity Modifying Protein 2) RAMP2. Control samples include both non-transfected cells and cells transfected with empty vector. There are three technical replications for each condition. RNA was extracted after 24 h, checked for quality and RNA integrity and profiled with whole exome RNA sequencing. Overall design: HEK293T cells were cultured in cell medium and 300,000 cells were seeded onto poly-D-lysine mol wt 70,000-150,000 (Sigma-Aldrich) coated cover glasses (18-mm, thickness No. 1.5H, Marienfeld Superior) placed in 60-mm tissue culture dishes. The next day, cells were transfected with 2 µg plasmid DNA for RNA sequencing, using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer's protocol. Plasmid DNA included RAMP2 with a FLAG-tag epitope sequence following the signal peptide, OLLAS-epitope, tag on the C-terminus in pcDNA3.1, codon optimized for expression in human cell lines. After 24 h incubation at 37°C, RNA was extracted using the miRneasy kit (QIAGEN) for RNA sequencing. Extracted RNA samples were examined with Bioanalyzer for RNA integrity and concentration before sequencing. RNA integrity number was 9.62 with 0.98 standard deviation. 100 ng of total RNA was used to generate RNA-Seq libraries using Illumina TruSeq stranded mRNA LT kit (Cat# RS-122-2101).   Libraries prepared with unique barcodes were pooled at equal molar ratios. The pool was denatured and sequenced on Illumina NextSeq 500 sequencer using high output V2 reagents and NextSeq Control Software v1.4 to generate 75 bp single reads, following manufactures protocol (Cat# 15048776 Rev.E). For read alignment after adaptor cutting we used STAR software. Quality control was verified with qualilmap.