Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Cells were loaded onto Integrated Fluidic Circuit chips (IFC; Fluidigm). We identified capture of multiple cells and empty wells using brightfield illumination, with validation of single cell capture also carried out using a genetically-encoded red fluorescent nuclear marker. Cell lysis, reverse transcription and cDNA pre-amplification were performed in the C1 Single-Cell Auto Prep IFC using the SMARTer PCR cDNA Synthesis Kit (Clontech) and the Advantage 2 PCR Kit, as specified by the manufacturer (protocol 100-7168 A2). ERCC RNA spike-in control mix (92 transcripts; ThermoFisher) was added to the chambers at a 1:1000 ratio. cDNA was harvested and the libraries were prepared using the Nextera XT DNA Sample Preparation Kit and the Nextera Index Kit (Illumina), according to the manufacturer's recommendations (protocol 100-7168 A2). Libraries from one chip were pooled, and paired-end 75bp sequencing was performed on 4 lanes of an Illumina NextSeq500.