Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: mES or neural precursor cells were taken directly from culture, trypsinized, washed in PBS, spun down and resuspended at a concentration of 3 x 10^5 cell per mL of complete media, mixed 7:3 with C1 suspension reagent (Fluidigm), and loaded onto C1 Single-Cell Auto Prep chips (C1 chips; Fluidigm). After loading, each of the cell isolation chambers on the C1 chip was optically inspected for the presence of a cell. Subsequently, these cells were lysed and SMART-Seq whole transcriptome amplified (WTA) products were prepared with the C1 System using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) and protocols provided by Fluidigm (full details available at www.fluidigm.com). WTA products were harvested from the C1 chip, diluted to a concentration of 0.15 ng/μL, and cDNA libraries were prepared using Nextera XT DNA Sample preparation reagents (Illumina) as per the manufacturer’s recommendations, with minor modifications. Specifically, reactions were run at ¼ the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30s to 60s. After the PCR step, all 96 samples were pooled without library normalization, cleaned twice with 0.9x AMPure XP SPRI beads (Beckman Coulter), and eluted in buffer TE. The pooled libraries were quantified using Quant-IT DNA High-Sensitivity Assay Kit (Invitrogen) and examined using a high-sensitivity DNA chip (Agilent). Finally, samples were sequenced deeply using either a HiSeq 2000 or a HiSeq 2500 sequencer (25 bp paired-end reads).