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[…] Total RNA was prepared using Trizol reagent (Invitrogen) according to manufacturer’s protocol (for limb buds, dissected anterior, posterior, distal, and proximal tissue was dissociated into single cell suspensions in Trizol using a syringe fitted with a 25G (0.5 mm) needle (Sigma Aldrich, BD Microlance), followed by acid phenol:chloroform:isopropyl alcohol extraction, and then digested with 2U DNaseI (ThermoFisher Scientific, Ambion) for 30 min at 37°C). RNA was reverse-transcribed to complementary DNA (cDNA) using QuantiTect Reverse Transcription Kit (Qiagen). The quantitative real-time PCRs were performed in a 7300 system (ThermoFisher Scientific, Applied Biosystems, Life Technologies) by using LightCycler 480 SYBR Green I Master (Roche) and gene specific primer sets for shh, Gabpα, and Etv4. The cycle threshold (CT) values from all quantitative real-time PCR experiments were analysed using ΔΔCT method. Data were normalized to GAPDH and expressed as fold changes over that in control treatment group. From each biological replicate three technical replicates were analysed. All statistical analyses were performed using a two-tailed Student’s t-test.RNA sequencing was conducted by GATC Biotech (Konstantz, Germany). Samples were only submitted with an OD 260/280 ratio ≥1.8, a 260/230 ratio ≥1.7, and a RNA Integrity Number value ≥8 as detected by Agilent Technologies 2100 Bionalayser. The InView Transcriptome Explore service provided by GATC was used to provide a randomly primed and amplified cDNA library with Illumina adaptors ready for sequencing. Illumina sequencing was conducted producing 50 bp single end reads and a guarantee of over 30 million reads per sample. All samples were analysed on the main Galaxy server by first checking sequence quality by FastQC. Reads were then trimmed and any Illumina sequencing adaptors removed as appropriate and aligned to the mouse genome (mm9, NCBI 37) using Tophat2 (Galaxy Tool Version 0.9). The results for each condition were fed into Cuffdiff (Galaxy Tool Version 2.2.1.3) and visualized using the R Bioconductor package CummeRbund (Release 3.2). This process was repeated using RNA isolated from both immortalized limb cell lines and isolated limb tissue. Two biological replicates were analysed for each condition. […]

Pipeline specifications

Software tools FastQC, TopHat, Cufflinks, CummeRbund
Applications RNA-seq analysis, Transcriptome data visualization
Organisms Mus musculus