title: GSM1139276: testes normal; Equus caballus; RNA-Seq
library_construction_protocol: Total RNA was isolated from the eight samples using variations of guanidinium-based protocols. RNA was purified from the articular cartilage samples as described in MacLeod et al. 1996 (PMID 8702559), from control synovial membrane, cerebellum, testis and placenta villous using acid guanidinium thiocyanate/phenol/chloroform extraction with alcohol and salt precipitations (Chomczynski & Sacchi 1987, PMID 2440339), and from LPS-stimulated synovial membrane and whole embryo with RNeasy mini kits (Cat# 74104; Qiagen). All samples were processed with a final RNeasy (Qiagen) silica gel spin column clean up, ethanol precipitation and solubilization in nuclease-free water (Cat# AM9937; Ambion). Quantitative and qualitative parameters of the RNA preparations were assessed using a NanoDrop ND-1000 and Bioanalyzer 2100 (Agilent, Eukaryotic Total RNA Nano Series II). All samples had 260/280 ratios ≥1.93, 260/230 ratios ≥1.75 and an Agilent RNA integrity number (RIN) ≥8.1. Library preparation and subsequent nucleotide sequencing was performed according to Illumina’s standard mRNA-seq kit protocol (Transcriptome Analysis: mRNA-seq, http://www.illumina.com/pages.ilmn?D=291). Messenger RNA was selected with PolyT capture beads from 3.5 to 10.0 μg of total RNA and fragmented by zinc metal ion hydrolysis. RNA fragments were converted into cDNA using random hexamer primers and made double stranded using RNase H and DNA polymerase. The cDNA fragments were ligated to sequencing adaptors using Illumina’s Genomic DNA sample prep. Fragments between 100 and 300 bp were size selected by gel electrophoresis, applied to sequencing flow cells and enriched by PCR to grow fragment clusters.
attributes.tag: GEO Accession
attributes.value: GSM1139276