GSM3965050: E185_NBN_43; Mus musculus; RNA-Seq
GFP positive cells were FAC-sorted from Hes5::GFP and Tbr2::GFP transgenic lines. A time course was performed with NSCs, BPs and NBNs isolated at almost alternate time points during development E10.5 to postnatal day 3 (P3). Single cell capture, lysis and cDNA preparation were performed with the Fluidigm C1 system. Cells were loaded on a microfluidic C1 Single Cell Auto Prep Array for mRNA Seq (5-10µm), and capture efficiency evaluated using microscopy. Lysis, reverse transcription and cDNA amplification were performed with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech/Takara) according to Fluidigm's guidelines for single-cell RNA-seq on the C1 system. cDNA was harvested, profiles checked on the Fragment Analyzer (AATI) and their concentration determined using Quant-iT PicoGreen dsDNA Assay Kit. For subsequent library preparation using Nextera XT DNA library preparation kit (Illumina) following the Fluidigm manual, cDNAs were normalized to 0.3 ng/µl. Libraries were pooled and sequenced SR75 on an Illumina NextSeq 500 system (75 cycles High Output v2 kit).