title: Illumina HiSeq 2500 paired end sequencing; RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids
design: RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids
library_construction_protocol: 3D in vitro models of patient derived colon cancer tumors were processed to single cells, labelled with PKH26 dye and separated by FACS into PKH26 Negative and PKH26 Positive cell fractions after 12 days in culture. Colon cancer PDOs were cultured in growth factor reduced Matrigel as previously described (Regan, J. L. et al. Non-Canonical Hedgehog Signaling Is a Positive Regulator of the WNT Pathway and Is Required for the Survival of Colon Cancer Stem Cells. Cell Rep. 21, 2813–2828 (2017). Cells were lysed in RLT buffer and processed for RNA using the RNeasy Mini Plus RNA extraction kit (Qiagen) Samples were processed using NuGEN's Ovation RNA-Seq System V2