GSM3097148: mature zone 2; Mus musculus; RNA-Seq
Laser capture microdissection (LCM) protocol: LCM was performed using the PALM Microbeam system (Zeiss). The articular zone, mature zone and hypertrophic zone were readily identified on the basis of cell morphology under 10×microscope objective. A focused laser beam was activated to dissect the zone of interestinto Adhesive Cap tube caps (Zeiss). A total of 25-30 sections from each zone were microdissected and pooled to create single samples. A total of three independent biological replicates were tested. The collected cell samples were immediately lysed in 50-μl Arcturus PicoPure RNA extraction buffer at 42°C for 30 min. Total RNA was extracted from cell lysates using Arcturus PicoPure RNA Isolation Kit (Applied Biosystems). To evaluate RNA quality, the RNA 6000 Pico kit and Bio-Analyzer 2100 (Agilent Biotechnologies) were used. RNA integrity number (RIN) ranging from 10 (intact) to 1 (totally degraded) was analyzed and only high-quality RNA samples (RIN value>6) were selected for RNA-sequencing. RNA samples were subjected to RNA-sequencing at the University of Rochester Genomics Research Center. Briefly, total RNAs from each sample were polyA selected and single-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit (Illumina).
GEO Accession
GSM3097148