title: GSM3731672: M2818; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Flash frozen epiphyseal tissue was pulverized using a mortar and pestle, and RNA was extracted using serial TRIzol (Ambion; Austin, TX)-chloroform extractions, with a modification to the standard protocol that we previously reported in the RNA precipitation step to account for the high GAG content in cartilage that can co-precipitate with RNA. Briefly, 100% isopropanol and a solution of 1.2 M NaCl and 0.8 M sodium citrate (Sigma-Aldrich; St. Louis, MO) in ddH2O was used in a 1:1 mixture to separate contaminating GAGs from the RNA pellet. Following precipitation, extracted RNA was in-column treated with RNase-free DNase (Qiagen; Valencia, CA) on miRNeasy columns (Qiagen; Valencia, CA) and eluted following the manufacturer's protocols. The quality of each RNA sample was verified via BioAnalyzer using the Agilent RNA 6000 Pico Kit (Agilent; Santa Clara, CA), to ensure all samples used for library preparation had an RNA Integrity Number (RIN) value > 7. High quality total RNA from each sample was used to prepare RNA-Seq libraries using the TruSeq mRNA stranded kit (Illumina; San Diego, CA). Paired-end, 100-base pair sequencing was performed using the Illumina HiSeq 2500 with the results mapped to the canine genome version CanFam3.1 (NCBI) using Ensembl build 77. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA; Qiagen, Valencia, CA, USA).
attributes.tag: GEO Accession
attributes.value: GSM3731672