Time-course RNA-seq in developing limb bud mesenchyme cells in mice
To analyse the digit formation process in mouse limb buds, we performed a time-course RNA-seq analysis across 6 stages of mouse hindlimb autopods from Sox9-EGFP mice. After cutting the autopod regions, ectoderm was removed, and mesenchyme cells were dissociated, followed by FACS sorting for Sox9-positive and Sox9-negative cells. We prepared cDNA libraries for both populations and also prepared a cDNA library from left and right autopods from the same embryo separately, serving as biological duplicates at each stage. cDNA library was prepared with SMARTer Ultra Low RNA Kit v3 for Illumina Sequencing (Clonetech) and NEBNext Ultra DNA Library Prep kit for Illumina (NEB) from 10ng of total RNA for each sample. Sequencing was done with HiSeq2500 (Illumina). We read 50bp with the paired end mode.