title: ESC_c2
organism: Mus musculus
description: Protocols: ESCs were cultured using standard procedures in medium containing 1,000U/ml leukemia inhibitory factor (Millipore) with irradiated MEF feeders (Global Stem) on gelatin-coated plates. ESCs were removed from feeder cells by soaking twice on gelatin-coated plates for 1 hour each prior to sample collection or cell differentiation. Differentiation was performed by culturing ESCs in the absence of MEF feeders in 50:50 DMEM/F12:Neurobasal medium (Gibco) supplemented with 1x N2 (Gibco), 1x B27 (Gibco), 40mg/L insulin (Sigma), 25ug/ml BSA Fraction V (Gibco) at 0.5x106-2.0x106 cells/10cm plate for 6 days. Cells were then resuspended in N2 expansion medium (DMEM/F12, 50ug/ml BSA fraction V, 10ng/ml EGF (Preprotech), 10ng/ml FGF (Preprotech), 1ug/ml laminin (Invitrogen), 1x N2) and plated onto uncoated T75 flasks to allow for neurosphere outgrowth. Following 4 days, neurospheres were collected by mild centrifugation and plated onto gelatin-coated plates in N2 expansion medium. Following 2-3 passages, cells represented a homogeneous population of NPCs, which could be seeded to expand single-cell derived clones. RNA from was isolated using Trizol reagent (Ambion) according to the manufacturer's instructions. PolyA+ RNA was isolated (Oligotex kit, Qiagen) from 10ug total RNA. Stranded libraries were prepared using a protocol adapted from (Parkhomchuk et al., 2009) for paired-end sequencing on the Illumina GA IIx platform.