characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-07-02T17:03:56Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:31:57Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2803906
characteristics.characteristic: External Id
characteristics.text: European Molecular Biology Laboratory - European Bioinformatics Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2015-07-02T17:03:56Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:31:57Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS560817
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2988:dFKHnls_1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: BALB/c CD4+CD25- T cells were isolated by negatively depleting splenocytes stained with FITC-conjugated depletion cocktail antibodies including anti-CD8, anti-CD19, anti-CD11b, anti-CD11c, anti-Ly6G, anti-TER119 and anti-CD25 using autoMACS machine (Miltenyi Biotc). Isolated mouse primary T cells were cultured in RPMI-1640 medium supplemented with 10% FCS, antibiotics and 50uM 2-mercaptoethanol. Primary T cells were transduced with virus carrying Foxp3 and its mutants. Transduced cells were allowed to rest for 48 h in RPMI medium with IL2. The resting cell expressing transduction marker rCD8a were sorted by FACS or autoMACS. For re-activation sample, sorted cells were activated with plate-coated anti-CD3 (5 ug/ml), soluble anti-CD28 (1 ug/ml) for 8 h. mRNA isolation was performed using uMACS mRNA isolation kit (Miltenyi Biotec). 5x106 cells of each sample were lysed for mRNA isolation. cDNA synthesis was performed using the uMACS cDNA synthesis module (Miltenyi Biotec). Second strand was synthesized using E.coli DNA polymerase I (NEB), E.coli DNA ligase (NEB) and E.coli RNaseH (NEB) in second strand synthesis buffer (Invitrogen) at 16C for 2.5 h. Double strand DNA was fragmented by sonication using Bioruptor UCD-400 (Diagenode), with 20 cycles of (30 sec On; 30 second off). Fragmented samples were end-repaired using Klenow large fragment (NEB), T4 DNA polymerase(NEB) and T4 polynucleotide kinase (NEB) at 20C for 30 min. Purified blunt-ended DNA was 3' adenylated using 3' greater than 5' exo- Klenow fragment (NEB) at 37C for 30 min. After purification, adapters containing 6 bp barcodes from sample prep oligonucleotide kit (Illumina) were ligated to 3' A-tailed DNA fragment at 20C for 30 min using T4 DNA ligase (NEB). Each DNA sample was amplified via 18-cycle PCR using universal primers from the sample prep oligonucleotide kit. The amplified library was size-selected from 200-400 bps using 2% agarose gel and quantified with a high-sensitivity DNA chip (Agilent) using agilent bioanalyzer.
characteristics.characteristic: description
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: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: dFKHnls_1
characteristics.characteristic: title
characteristics.text: Foxp3 delta-FKHnls
characteristics.characteristic: transduced gene