title: Illumina HiSeq 4000 paired end sequencing; Single-cell RNA-seq of  motor neuron progenitors differentiated from mouse embryonic stem cell line HM1 by treatment with RA and SAG
design: Single-cell RNA-seq of  motor neuron progenitors differentiated from mouse embryonic stem cell line HM1 by treatment with RA and SAG
library_construction_protocol: NPs were differentiated as described previously (Gouti et al. 2014). In brief, HM1 (Thermo Scientific) ESCs were maintained in ES cell medium with 1000 U/ml LIF on mitotically inactivated mouse embryonic fibroblasts (feeder cells). For differentiation cells were dissociated in 0.05% Trypsin (Gibco) and replated onto tissue culture plates for 25 minutes to remove feeder cells. Cells staying in the supernatant were spun down and resuspended in N2B27 medium at a concentration of 106 cells / ml. 45000 cells were plated onto 35mm CellBind dishes (Corning) precoated with 0.1% Gelatine solution in 1.5 ml N2B27 + 10 ng / ml bFGF. At D2 medium was replaced with N2B27 + 10 ng/ml bFGF + 5 µM CHIR99021 (Axon). At D3, and every 24 hours afterwards, medium was replaced with N2B27 + 100 nM RA (Sigma) + 500 nM SAG (Calbiochem). Single-cell RNA extractions were performed on the C1 Single-cell Auto Prep integrated Fluidic Circuit (IFC) according to the manufacturer's protocol (PN 100-7168, Fluidigm) Single cell cDNA was generated using the Clontech SMARTer Ultra Low RNA kit on the Fluidigm C1 chip. ArrayControl RNA Spikes were added to the cell lysis mix as recommended in the Fluidigm protocol. Libraries were prepared using the Illumina Nextera XT DNA Sample Preparation kit.