GSM1642481: Adult_Hip_fluox1_RNAseq; Rattus norvegicus; RNA-Seq
RNA extraction was carried out using RNeasy lipid tissue kit (QIAGEN, Venlo, The Netherlands) Total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN) and fragmentation by hydrolysis (5× fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s. Fragmented mRNA was purified (RNeasy MinElute Kit; QIAGEN) and used for cDNA synthesis with 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen Life technologies, Bleiswijk, The Netherlands). Double stranded cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer's recommendations and purified using the Minelute Reaction Cleanup Kit (QIAGEN) according to the manufacturer's protocol. DNA samples were prepared for RNA-seq by end repair of 20 ng DNA as measured by Qubit dsDNA HS (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Quality control of DNA libraries prepared for sequencing was made by qPCR and by running the products on a Bioanalyzer (Bio-Rad, Veenendaal, The Netherlands). Cluster generation and sequencing (36 bp, single read) was performed with the Illumina Genome Analyzer IIx (GAIIx) platform according to standard Illumina protocols. Samples were sequenced to a depth of approximately 15 million uniquely mapped tags per sample. Sequences were aligned to the rat rn4 reference genome with the Illumina Analysis Pipeline allowing one mismatch. Only the tags aligning to one position on the genome were considered for further analysis. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing.