characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-05-31T17:02:00Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-03-23T21:09:33Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA103939387
characteristics.characteristic: External Id
characteristics.text: Cancer Research UK Cambridge Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2017-05-31T17:02:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-03-23T21:09:33Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1628555
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5522:20547_8#31
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Trophoblast stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000351
characteristics.characteristic: cell line
characteristics.text: trophoblast stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000351
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Murine TSCs were kindly provided by Dr. Jennifer Nichols. TSCs were cultured on mouse embryonic fibroblast (MEF) feeders with TSC culturing medium (a combination of 70% MEF conditioned media (R&D systems) and 30% RPMI 1640, supplemented with 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 uM B-mercaptoethanol, 25 ng/mL human recombinant FGF4 (R&D systems) and 1 ug/mL heparin (Tocris Bioscience)). To prepare for single-cell sorting, cells were harvested with trypsin and MEF feeders were depleted by plating the cells onto a gelatinised plate followed by incubation for 1h at 37 degrees C on TSC culturing medium. The supernatant containing TSCs was used for sorting. Reverse transcription was performed in a final volume of 13.2 ul per well, containing 1 uM of oligo-dT (Sigma-Aldrich), 1.04 mM of each dNTP (ThermoFisher), 100 U of SuperScript II retrotranscriptase (Invitrogen/ThermoFisher), 5 U of RNase inhibitor (Ambion), 5 mM of DTT, 1 M of Betaine (Sigma-Alrich), 6 mM of MgCl$_2$ (Ambion) and 1 uM of TSO primer (Exiqon). Spike-in RNA was added such that each well contained a final volume of 0.1 ul of a 1:3,000,000 dilution of the ERCC RNA Spike-In Mix (Invitrogen/ThermoFisher) and 0.12 ul of a 1:3,000,000 dilution of the Spike-in RNA Variant (SIRV) Control Mix E0 (Lexogen). Cells were sorted into individual wells of a 96-well microtiter plate. Each well contained 2.3 ul of lysis buffer with RNAse inhibitor (Ambion) in a 0.2% (v/v) Triton X-100 solution. Preamplification was performed in a total volume of 27 ul that contained 13.5 ul of HiFi Hotstart ReadyMix (2x; KAPA Biosystems) and 0.1 uM of IS PCR primer (Sigma-Aldrich). After 23 cycles of amplification, samples were cleaned with 80% (v/v) of Ampure beads (Beckman Coulter). Sequencing libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina).
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: wild type phenotype
characteristics.characteristic: phenotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: OK
characteristics.characteristic: single cell well quality
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: 20547_8#31
characteristics.characteristic: title