GSM7048037: Adultplts_3; Mus musculus; RNA-Seq
Trizol RNA extraction To remove contaminating DNA and DNases, 600 ng of RNA from each sample was processed with the TURBO DNA-free kit (Invitrogen) according to manufacturers instructions. Cleaned RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA and amplified with PCR primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina flow cell and sequenced using the NextSeq 550 sequencer (Illumina, San Diego, CA). Single end reads of 75nt were generated for each sample.