Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids

ABSTRACT: 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 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).

INSTRUMENT(S): BD FACSARIA II, Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Joseph Regan

PROVIDER: E-MTAB-8924 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We sought to determine which gene transcripts are enriched in Wnt-responsive adrenocortical mouse cells compared to the entire adrenocortical mouse cell population in vivo. To this end, we employed transgenic reporter mice that label Wnt-responsive cells with GFP expression (TCF/Lef:H2B-GFP mice) or label all adrenocortical cells with GFP expression (Sf1:eGFP mice). GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genotype. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267). One TCF/Lef:H2B-GFP array was deemed low-quality and discarded. Two-sample T-tests were used to compare the two groups of samples. We also supply a supplementary file holding the data and some statistical analysis, as well as probe-set annotation that we used at that time (users may wish to obtain new annotation though). We analyzed only 28944 probe-sets with category "main", "---", and "flmrna->unmapped" according to Affymetrix annotation. GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genoytpe. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267).

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RNA-sequencing of cycling PKH26-Negative cells and PKH26-Positive label retaining quiescent cancer stem cells from colon cancer patient-derived organoids

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