PROTOCOLS

Transcriptional profiling

The poly-adenylated transcript abundance of whole E9.5 embryos is measured for embryonic lethal lines and wild-types, by high-throughput transcriptome sequencing.

Sample preparation

Whole embryos are dissected and, after counting the number of somites, stored in RNAlater at -20 °C.

The embryos are mechanically lysed in TRIzol using a 5 mm steel bead and a Qiagen TissueLyser set at 20 Hertz for 2 minutes. Following the addition of chloroform, a 15 minute room temperature incubation and centrifugation, the nucleic acid is collected from the clear top aqueous phase into a clean 1.5 ml tube.

Nucleic acid is DNase treated (NEB) for 10 minutes at 37 °C to remove contaminating DNA. Samples are cleaned using RNeasy MinElute clean-up kit (Qiagen) under the standard protocol and quantified using Qubit RNA BR Assay Kit (Invitrogen).

Sequencing

Stranded RNA-seq libraries are constructed using the Illumina TruSeq Stranded RNA protocol (dUTP method) with oligo dT pulldown. Libraries are sequenced using Illumina HiSeq2000 to 75 bases with paired end reads.

Transcriptional profiling is performed by the Vertebrate Genetics and Genomics group at the Wellcome Trust Sanger Institute.