GSM5335333: R3: Articular cartilage right leg rep3 week 12; Mus musculus; RNA-Seq
Total RNA was isolated using TRIzol followed by RNeasy mini RNA isolation kit (Qiagen) and DNase (Qiagen) treatment. RNA-Seq library preparations were done using established Illumina methods for mRNA-Seq (Part #RS-122-2001). Briefly, poly A+ RNA was purified from 100 ng of total RNA with oligo-dT beads and fragmented with divalent cations at elevated temperature. First strand cDNA synthesis was performed with random hexamer priming and SuperScript II Reverse transcriptase (Invitrogen, part # 18064-014). Second strand cDNA synthesis was performed using RNAseH and DNA PolI. Following dscDNA synthesis, the double stranded products are end repaired, followed by addition of a single 'A' base and then ligation of the Illumina TruSeq adaptors. The resulting product was amplified with 15 cycles of PCR. Each library was made with a unique Index sequence and libraries are pooled for sequencing.