GSM9786072: LLC Sildenafil-3; Mus musculus; RNA-Seq
Following their respective treatment durations, cells were washed with phosphate-buffered saline (PBS) and lysed using RLT buffer. Total RNA was subsequently isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions mRNA library preparation, we used the Illumina TruSeq stranded mRNA ligation kit. We use between 100ng to 1ug of total RNA as the input to an mRNA capture with oligo-dT coated magnetic beads. The mRNA is fragmented, and then a random-primed cDNA synthesis is performed. The resulting double-strand cDNA is used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification being performed to give you a sequencing ready library. The final purified product is then quantitated by qPCR before cluster generation and paired end ( 2x100 cycle) sequencing on NovaSeq.