normalisation data transformation protocol
The Tophat aligner (http://tophat.cbcb.umd.edu/) was used to align the reads to the mouse reference genome (mm9). After alignment the read counts for each gene were extracted using htseq-count (http://www-huber.embl.de/users/anders/HTSeq/) based on an mm9 Refseq gff file. Differential expression in our two groups was evaluated using DESeq version 1.4.1, implemented in R 2.14.1. DESeq uses a negative binomial distribution to model genic read counts following normalisation based on size factors and variance. As for the microarray analysis, p-values were adjusted by the procedure of Benjamini and Hochberg to control the type I error rate, and a cut off of p less than or equal to 0.05, and a fold change of greater than or equal to 2 were used as a threshold to define differential expression.
nucleic acid sequencing protocol
Sequencing on the Genome Analyzer 2 (Illumina). 40bp single end reads were obtained from an Illumina GAII in FASTQ format, one sample per sequencing lane.
Heterozygous matings of Splotch-delayed mice were harvested at E14.5 (TS23)
nucleic acid extraction protocol
RNA was extracted following manufacturers guidlines ( SV. Total RNA Isolation system, Promega, UK)
nucleic acid library construction protocol
The DNase-treated RNA (3ug) was used to prepare RNA-Seq libraries with the TruSeq RNA Sample Prep kit. A total of six cDNA libraries were constructed, representing triplicate biological replicates for each group.
Splotch-delayed homozogous mutant and control (homozygous wild-type and heterozygote) embryos were dissected and Humerus forelimb tissue was dissected at embronic day 14.5 (Theiler Stage 23)