E-MTAB-8924
RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids
Recent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) are the source of relapse following treatment. The isolation and molecular characterisation of qCSCs may therefore lead to the identification of novel therapeutic targets for their elimination.  Label retention of the lipophilic fluorescent dye PKH26, wherein dividing cells lose the label and quiescent or slow cycling cells retain the label for an extended period of time, was used to isolate quiescent cancer stem cells (qCSCs) from a panel of colon cancer patient-derived organoids (PDOs).   PDOs were dissociated to single cells, labelled with PKH26 and plated in Matrigel culture. After 12 days, PDOs were processed to single cells, labelled with DAPI (to exclude dead cells) and isolated by fluorescence assisted cell sorting (FACS) into two fractions of PKH26-Negative and PKH26-Positive (label retaining) cells. Functional analyses of these cellular subpopulations demonstrated that PKH26-Positive cells are self-renewing qCSCs.   RNA was collected from FAC sorted PKH26-Negative and PKH26-Positive cells and analysed by RNA-sequencing.  Cells were lysed in RLTplus buffer and RNA was extracted using Qiagen RNeasy Mini Kit. RNA quality was assessed prior to sequencing.  Samples were processed using NuGEN's Ovation RNA-Seq System V2 and Ultralow V2 Library System and sequenced on an Illumina HiSeq 2500 machine as 2x125nt paired-end reads.   Reads were mapped to the human reference genome (assembly hg19) using the STAR aligner (version 2.4.2a). Total read counts per gene were computed using the program “featureCounts” (version 1.4.6-p2) in the “subread” package, with the gene annotation taken from Gencode (version 19).
Recent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) are the source of relapse following treatment. The isolation and molecular characterisation of qCSCs may therefore lead to the identification of novel therapeutic targets for their elimination.  Label retention of the lipophilic fluorescent dye PKH26, wherein dividing cells lose the label and quiescent or slow cycling cells retain the label for an extended period of time, was used to isolate quiescent cancer stem cells (qCSCs) from a panel of colon cancer patient-derived organoids (PDOs).   PDOs were dissociated to single cells, labelled with PKH26 and plated in Matrigel culture. After 12 days, PDOs were processed to single cells, labelled with DAPI (to exclude dead cells) and isolated by fluorescence assisted cell sorting (FACS) into two fractions of PKH26-Negative and PKH26-Positive (label retaining) cells. Functional analyses of these cellular subpopulations demonstrated that PKH26-Positive cells are self-renewing qCSCs.   RNA was collected from FAC sorted PKH26-Negative and PKH26-Positive cells and analysed by RNA-sequencing.  Cells were lysed in RLTplus buffer and RNA was extracted using Qiagen RNeasy Mini Kit. RNA quality was assessed prior to sequencing.  Samples were processed using NuGEN's Ovation RNA-Seq System V2 and Ultralow V2 Library System and sequenced on an Illumina HiSeq 2500 machine as 2x125nt paired-end reads.   Reads were mapped to the human reference genome (assembly hg19) using the STAR aligner (version 2.4.2a). Total read counts per gene were computed using the program “featureCounts” (version 1.4.6-p2) in the “subread” package, with the gene annotation taken from Gencode (version 19).