Illumina HiSeq 4000 sequencing; RNAseq of mouse presomitic mesoderm segments along the anteroposterior embryo axis.
RNAseq of mouse presomitic mesoderm segments along the anteroposterior embryo axis.
B6 pre-turned embryos were collected and dissected in M2 media. The mesoderm was separated from the other germ layers after treatment with pancreatin for one minute at 37° C. The left and right sides of the presomitic mesoderm were finely dissected using tungsten needles, and each was cut into five segments along the anteroposterior axis. Each segment was collected in 15µl of lysis buffer (0.2% Triton X100 plus 1:20 RNase inhibitor (Clonetech)) that had been prepared fresh at the start of the dissections. Samples were vortexed, centrifuged and frozen on dry ice. Samples were processed with the Smart-seq2 protocol as described in Picelli et al. Nature Protocols, 2014. doi:10.1038/nprot.2014.006 Libraries were prepared using the Illumina Nextera XT DNA preparation kit.