Nextera xt library prep system

We prepared sequencing libraries as previously reported^{19 (link)}. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 19 or 22 cycles for RNA from single hESC or cortical progenitor cells, respectively. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100, or with the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). Single cells from primary tissue contained a lower amount of mRNA compared to H1 hESCs, requiring a reduction in ERCC spike-in RNAs by 10-fold and addition of three extra PCR cycles. We carried out sequencing of human cortical progenitors on Illumina MiSeq using 31 base paired-end reads. We carried out sequencing of hESCs on the HiSeq using 50 base paired-end reads. We saw few global differences in read statistics between MiSeq runs and HiSeq runs when samples were assessed on both instruments.