characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-21T16:21:53Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-21T16:21:53Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112225206
characteristics.characteristic: External Id
characteristics.text: IRBBarcelona
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-21T16:21:53Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-21T16:21:53Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14336046
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12412:Sample1_library2
characteristics.characteristic: Submitter Id
characteristics.text: 54
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: A375, BRAF mutant-A375 isogenic cell line
characteristics.characteristic: cell_line
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were cultured in 75cm2 flasks with DMEM 10%FCS 1% penicillin/streptomycin at 37°C/5%CO2. A375 parental cells remained untreated. A375-MAPKi resistant cells, A375-etomoxir cells and A375-ranolazine cells had been made resistant to vemurafenib by treating the cells with 5 uM vemurafenib for one week followed by continuous culture under 0.5 uM vemurafenib until ressitant colonies arose. All these 3 cell lines were cultured with 0.5 μM vemurafenib up until cell processing for scRNAseq. A375-etomoxir cells had been selected with 100 μM etomoxir during acquisition of resistance to vemurafenib, but no longer after that. A375-ranolazine cells had been previously selected with 100 μM ranolazine during acquisition of resistance to vemurafenib, but no longer after that. Cells were trypsinised and resuspended in fresh medium before being passed through a cell strainer to obtain a single-cell suspension. Samples were centrifuged again for at 300g 5 min 4°C , resuspended in FACS buffer (sterile-filtered 1% FCS, 2mM EDTA, 25mM HEPES in 1X PBS). Following, incubation with 1ug/mL DAPI for 30 min on ice and darkness, samples were washed with FACS buffer, centrifuged at 300g 4°C 5 min twice. FACS isolation was issued gating DAPI negative population (live cells) and double discrimination of singles and doublets. Then cells were sorted into individual tubes per group condition, 15000 cells per group. Cell integrity and number was assessed by trypan blue and bright-field microscopy in every condition. Live cells were encapsulated intro droplets and libraries were prepared using Chromium Single Cell Reagents kit v3.1 according to the manufacturer’s protocol. RNA extraction is performed within single cell v3.1 gel beads as described by manufacturer 10X genomics. Reverse transcription is then procured within each sample and the results is GEMs with barcoded full-length cDNA from poly-adenylated mRNA.Immediately, following the Gel GEM generation, the Gel Bead is dissolved, primers are released, and any copartitioned cell is lysed. Primer containing: an Illumina TruSeq Read 1 (read 1 sequencing primer), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI) and 30 nt poly(dT). Sequence are mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA. For the generation of the libraries, the kit for Next GEM Single Cell 3’ Reagents Kits v3.1 (Dual Index) has been used, pooling four samples per library (labeling each sample with hashtag oligonucleotides). After incubation, GEMs are broken, and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes biochemical leftover reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: melanoma
characteristics.characteristic: disease
characteristics.text: BRAF V600E mutation
characteristics.characteristic: genotype
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: skin
characteristics.characteristic: organism part
characteristics.text: parental, vemurafenib-resistant
characteristics.characteristic: phenotype
characteristics.text: neoplasm
characteristics.characteristic: sampling site
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: Sample1_library2
characteristics.characteristic: title