GSM5605538: NA4; Homo sapiens; RNA-Seq
We used TRIzol (Invitrogen, Carlsbad, USA) to extract total RNA from each meniscus cell, following the manufacturer's protocol. The RNA quality was checked by an Agilent 2200 and kept at −80 °C, and only samples with a RIN (RNA integrity number) value >7.0 were used for the cDNA library construction. The cDNA libraries were constructed for each pooled RNA sample using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina according to the manufacturer's instructions. Generally, the protocol consists of the following steps: depletion of rRNA and fragmented into 150-200 bp using divalent cations at 94 ℃ for 8 min. We further used Dnase I to eradicate contamination after we wiped off ribosome RNA. The cleaved RNA fragments were reverse-transcribed into first-strand cDNA, second-strand cDNA synthesis, fragments were end repaired, A-tailed and ligated with indexed adapters. Target bands were harvested through AMPure XP Beads(beckman coulter) .The products were purified and enriched by PCR to create the final cDNA libraries and quantified by Agilent2200. The tagged cDNA libraries were pooled in equal ratio and used for 150 bp paired-end sequencing in a single lane of the Illumina HiSeqXTen.