biostudies-arrayexpressJiri NovotnyHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-9216Photo-damage represents an important aspect of cutaneous carcinogenesis. The photo-damaged microenvironment significantly facilitase progression of malignant melanoma. Therefore human primary dermal fibroblasts from photo-damaged adult skin and juvenile sun-protected skin were isolated and expanded as described by Dvorankova et al. (PMID: 29675784). Low passage fibroblasts were mixed in suspension (in ratio 1:1) with G361 malignant melanoma cell line (CVCL_1220). Using the hanging drop technique (PMID: 29786551), the melanoma/fibroblast spheres containing 50,000 cells per sphere were formed during the next 72 hours. The organoids were consequently maintained for another 2 days in non-adhesive culture wells in complete DMEM culture medium. At this time, heterogeneous spheres were harvested for immunohistochemical analysis or for further invasion studies. Single-cell suspensions from heterogeneous spheres were analysed by single-cell RNA sequencing (10x Chromium V3) to reveal the transcriptional heterogeneity of model cell populations under given conditions with emphasis on the features of dermal fibroblasts exposed to the influence of malignant melanoma.biostudies-arrayexpressSequencing - All single-cell RNA-sequencing was performed with an Illumina Nextseq 500 with the following setup: paired-end, read1 length 28bp, read2 length 56bp.Nucleic Acid Extraction - Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 4,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 4 rxns PN-1000092 was used.Library Construction - Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 4,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 4 rxns PN-1000092 was used.Growth Protocol - Cell suspensions were inoculated in DMEM + 10% fetal bovine serum for 72 hours in hanging drops. Heterogeneous spheres were cultured for additional 24 hours in non-adhesive culture dishes.Sample Collection - Heterogeneous spheres were prepared from primary human fibroblasts and G361 melanoma cell line (commercially available; CVCL_1220). Fibroblasts were isolated from photo-aged skin of an adult and from juvenile photo-protective skin of child.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw data from Illumina Nextseq 500 sequencer were processed by cellranger software (v3.1.0) provided by 10x Genomics. Cellranger mkfastq command was run on each sequencing run. Cellranger software (v3.1.0, GRCh38.93) provided by 10x Genomics was run (count command) on raw FASTQ files of each sample (both sequencing runs). Cellranger count pipeline output was provided for each sample (mtx format).Data Transformation - Raw feature barcode matrices were imported to Seurat v3 R package and saved in TSV format. TSV matrices were provided.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of coding RNA from single cellsHomo sapiensMichal KolarKarel SmetanaLukas LacinaJiri NovotnyfalseSingle-cell RNA-seq (10x Chromium V3) of heterogeneous spheres consisting of melanoma cell line and two types of human primary dermal fibroblasts.Photo-damage represents an important aspect of cutaneous carcinogenesis. The photo-damaged microenvironment significantly facilitase progression of malignant melanoma. Therefore human primary dermal fibroblasts from photo-damaged adult skin and juvenile sun-protected skin were isolated and expanded as described by Dvorankova et al. (PMID: 29675784). Low passage fibroblasts were mixed in suspension (in ratio 1:1) with G361 malignant melanoma cell line (CVCL_1220). Using the hanging drop technique (PMID: 29786551), the melanoma/fibroblast spheres containing 50,000 cells per sphere were formed during the next 72 hours. The organoids were consequently maintained for another 2 days in non-adhesive culture wells in complete DMEM culture medium. At this time, heterogeneous spheres were harvested for immunohistochemical analysis or for further invasion studies. Single-cell suspensions from heterogeneous spheres were analysed by single-cell RNA sequencing (10x Chromium V3) to reveal the transcriptional heterogeneity of model cell populations under given conditions with emphasis on the features of dermal fibroblasts exposed to the influence of malignant melanoma.2021-05-12T00:00:00Z2025-05-12T13:30:13.158Z2025-05-12T13:30:13.158ZE-MTAB-9216EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184