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