title: Illumina HiSeq 2500 sequencing; Ngn2 induces diverse neuronal lineages from human pluripotency
design: Ngn2 induces diverse neuronal lineages from human pluripotency
library_construction_protocol: Ngn2-iNs were selectively dissociated from co-cultured rat astrocytes using a mild dissociation procedure. Enriched neural cell suspensions were diluted to 500-1000 cells/mL. Single cells were encapsulated along with beads and reverse transcription reagents into droplets using 10X microfluidic chip system. Reverse transcription of first strand cDNA was performed in droplets. Induced pluripotent stem cells and corresponding rtTA/Ngn2-derivatives were cultured in standard feeder-free conditions in mTeSR1 (StemCell Technologies) on plates coated with matrigel (Corning). Primary cortical rat astrocytes (Gibco) were cultured in high-glucose DMEM containing 10% FCS and 1% Pen/Strep on plates coated with poly-D-Lysine (Sigma-Aldrich). Fresh media was added to the astrocytes every 4-5 days and passaged once a week with Trypsin-EDTA digestion at a standard ratio of 1:2. Astrocytes were used up to passage 10, with passage 0 being the culture of initial isolation. rtTA/Ngn2 double positive stem cell lines were generated and differentiated into Ngn2-iNs as previously described (Frega et al., 2017). Single stranded cDNA was cleaned up and amplified after breaking the droplets generated during the sample collection using the 10X 3' Single cell Kit v2 according to the manufacturer's instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay.  Libraries were constructed following the 10X library construction protocol v2. Briefly, cDNA was fragmented, end repaired and adaptor sequences were ligated, followed by a PCR to itroduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.