characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-29T12:20:28Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-29T12:20:28Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111492393
characteristics.characteristic: External Id
characteristics.text: IRB Barcelona / BIST
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-29T12:20:28Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-29T12:20:28Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13586404
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12315:PDL1_Rano
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: 6767
characteristics.characteristic: cell count
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Samples were obtained by dissecting the melanoma tumour from the animal skin, removing the complete skin from the tumour. Scissors and scalpel were used to remove the tissue and then it was placed in cold PBS on ice until tissue processing. A piece of 100 mg was weighed and chopped using sharp razors and scalpels in cold DMEM without FBS and P/S on ice. A final concentration of 0.25 mg/mL of Liberase DH (Merck, 54010544001), 0.55 mg/mL of Dispase II (Merck, 4942078001) and 150 U/mL of DNAse (Roche, 11284932001) was added in a total of 1 mL of solution. Samples were incubated at 37°C for 30 min and then homogenised with increasing gauge syringes up to 25G. Then, samples were centrifuged at 300g 4°C 5 min and 1X red-blood lysis buffer (Biolegend, 420301) was applied for 5 min RT and then filtered through a 40uM cell strainer. 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) and 1:50 TruStain FcX mouse antibody (Biolegend, 156604) for 10 min on ice and darkness. Following, incubation with anti-CD45-PE antibody (Biolegend, 103106) 1:100 and 1ug/mL DAPI for 30 min on ice and darkness. Finally, 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 CD45-PE+ 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 CD45-PE+ cells were encapsulated intro droplets and libraries were prepared using Chromium Single Cell Reagents kit v3.1 according to the manufacturer’s protocol Animals were culled humanely when they reached the maximum volume or when the experimental endpoint dictated. Tumours were removed from the animal skin, removing the complete skin from the tumour. Scissors and scalpel were used to remove the tissue and then it was placed in cold PBS on ice until tissue processing. Upon tumour resection, the samples were divided (whenever possible given the tumour size) for RNA extraction, whole tissue snap freeze and OCT tissue embedding and/or the different procedures described in the article. Mice were given subcutaneous injections of a BRAFV600mut melanoma cell line (5555) of 5 x 106 cells on the right flank of immunocompetent mice on day 0. Animals were monitored daily when tumour was evident and palpable (70 mm3) Around day 6 post injection (6DPI), animals were then divided into groups and treated accordingly. Ranolazine group was given 150 mg/kg intraperitoneal injection of Ranolazine (MedChemExpress, HY-17401) on day 6DPI and 300 mg/kg from 7DPI to end point. PD-L1 group was given 10mg/kg anti-PD-L1 (BioxCell, PD-L1 B7-H1, BE0101) starting on 6DPI and every 3 days for a total of 5 doses until end point. PD-L1 + Ranolazine group was given both treatments as described previously. Control group was injected with PBS daily until endpoint. Mice were sacrificed when tumours volume reached below 1500 mm3 and culled humanely. 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: [i] an Illumina TruSeq Read 1 (read 1 sequencing primer) [ii] 16 nt 10x Barcode [iii] 12 nt unique molecular identifier (UMI) [iv] 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 foe Next GEM Single Cell 3’ Reagents Kits v3.1 (Dual Index) has been used. 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: wild type genotype
characteristics.characteristic: genotype
characteristics.text: injected with 5555 BRAFv600 mutant mouse melanoma cells
characteristics.characteristic: growth condition
characteristics.text: PDL1_Rano 1
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: neoplasm
characteristics.characteristic: organism part
characteristics.text: Replicate 1
characteristics.characteristic: replicate
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: PDL1_Rano
characteristics.characteristic: title