title: Sequencing of subcutaneous vascular stem cells
design: For RNA library construction and deep sequencing, approximately 5 μg of RNA representing each group were submitted to Illumina/Solexa’s for sequencing. Sequence tags were prepared according to the Illumina/Solexa’s standard operation protocol. In brief, mRNA was isolated from 5μg total RNA by binding the mRNA to magnetic oligo bead. First- and second- strand cDNA were synthesized while the mRNA was attached to the beads. The double stranded cDNAs were digested with NlaIII. The 3’-cDNA fragments attached to the oligo(dT) beads were ligated to the Illumina GEX NlaIII Adapter 1, which contained a recognition site for the endonuclease MmeI for cutting 17 bp downstream of the recognition site (CATG) to produce tags with adapter 1. After removing 3’ fragments with magnetic beads precipitation, an Illumina GEX adapter 2 was introduced at the site of MmeI cleavage. The resulting adapter-ligated cDNA tags were amplified using PCR-primers that were annealed to the adaptor ends for 15 cycles. The 85 base fragments were purified and recovered by 6% polyacrylamide Tris–borate–EDTA gel. The purified cDNA tags were sequenced on the Illumina Cluster Station and Genome Analyzer. Image recognition and base calling were performed using the Illumina Pipeline.