Truseq rapid v2

E11 embryos were obtained from Runx1-GFP/+ mice mating and AGM cells stained with NG2 Cy3 (1:100, Millipore, ab5320c3), PDGFRβ APC (1:100, Biolegend, 136008), CD31 PECy7 (1:4000, eBioscience, 25-0311-82), CD45 PerCypCy5.5 (1:400, BD Pharmingen, 550994) and ckit BV421 (1:500, BD Horizon, 562609) for 30 min at 4 °C, washed, centrifuged, then resuspended in PBS/FCS/PS for analysis and cell sorting. Cells were purified using BD FACS ARIA Fusion cell sorter (BD Bioscience) and Sytox AAD was used to select viable cells. Cells were sorted and collected directly into 10–20 µl of lysis buffer containing Nuclease-free water (Ambion AM9930), 0.2% Triton and 1/20 RNAse inhibitor (Thermoscientific 10777019). Full-length cDNA was generated from 2.3 µl of this cell lysate using the Smarter2 procedure as described^{75 (link)}. Sequencing libraries were generated from 500 pg of cDNA with Illumina’s Nextera XT sample prep kit (Illumina Inc., U.S.A) and according to the Illumina TruSeq Rapid v2 protocol on the HiSeq2500 with a single read 51 bp and dual 9 + 9 bp index (Illumina Inc., U.S.A). Reads were aligned against the GRCm38 reference using HiSat2 (version 2.0.4). We called gene expression values using GENCODE M19 gene annotation file and the union mode in the Bioconductor Genome Alignments Package (v1.8.1).