Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: The single cell RNA-seq dataset is comprised of 15 separate drop-seq runs. For each run, a total of 2000 fully growth organoids were pooled, typsinized with TrypLE in presence of 10 µM Y-27632, strained into a single cell suspension and counted. The final concentration of the cell suspension varied between 100-120 cells/uL depending on the flow rate at which monodisperse droplets were formed. Organoids were always harvested for drop-seq analysis at same growth size to ensure uniformity from run to run. The droplets were broken and the beads were collected and reverse-transcribed. The cDNA obtained was then PCR amplified and quantified. Finally, the cDNA was fragmented
and amplified using primers that
allow amplification of only the 3'
ends, processed into RNA-seq
libraries using Illumina Nextera XT library prep kit. As described in Macosko et al Cell (2015)