ERC000011
ENA-CHECKLIST
2022-08-16T00:16:51Z
ENA-FIRST-PUBLIC
2022-08-16T00:16:51Z
ENA-LAST-UPDATE
SAMEA110464471
External Id
Genetech
INSDC center name
2022-08-16T00:16:51Z
INSDC first public
2022-08-16T00:16:51Z
INSDC last update
public
INSDC status
ERS12562497
SRA accession
E-MTAB-12028:WT
Submitter Id
ArrayExpress
broker name
fibroblast of dermis and leukocyte
cell_type
house mouse
common name
Protocols: For subcutaneous KPR tumors, KPR cells were trypsinized, filtered, counted, and resuspended in a 1:1 mixture of Hanks’s buffered saline solution and phenol-red-free Matrigel (Corning) at a concentration of 1x106 cells/ml. For all genotypes of mice used, age and sex-matched 6-12-week-old mice were inoculated subcutaneously in the right unilateral flank with 1x105 KPR tumor cells. Flank skin hair was shaved prior to implantation. Tumor volumes were measured and calculated 2-3 times per week using the modified ellipsoid formula: ½ × (length × width2). Tumors >1000mm3 were considered progressed and animals were removed from the study. Similarly, animals whose tumors ulcerated greater than 5mm were removed from study. From the remaining mice tissue was harvested 21 days post tumor implantation. Tumors were collected, weighed and minced into small pieces. To isolate naive flank skin, hair was shaved, adipose tissue was removed and skin tissue was minced. All tissues were subsequently enzymatically digested using a cocktail of dispase (Life Technologies, Carlsbad, CA), collagenase P and DNaseI (Roche, Penzberg, Germany) for 45 min at 37°C, to obtain a single cell suspension. Cells were counted using a Vi-CELL XR (Beckman Coulter, Brea, CA). For cytokine staining, cell suspensions were stimulated with eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (cat. 00-4975-93) resuspended in RPMI with 10% FBS plus 2 mmol/L l-glutamine and 2-Mercaptoethanol for 2 hours at 37°C.Cells were labelled with monoclonal antibodies purchased from BioLegend. Samples were enriched for CD24-CD45- stromal cells and CD45+ immune cells. Sorted cells were loaded onto a 10x Chromium Chip using reagents from the Chromium Single Cell 3’ Library Kit (10x Genomics) according to the manufacturer’s protocol. Amplified cDNA was used for 3’ RNA-seq library generation, RNA-seq libraries were prepared following the manufacturer’s user guide (10x Genomics).
description
adult
developmental stage
cancer
disease
wild type genotype
genotype
injected subcutaneous with KPR cells
growth condition
CD24-CD45- and CD45+
immunophenotype
Mus musculus
organism
Mus musculus
organism
skin of body
organism part
Mus musculus
scientific_name
WT
title