characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-03-01T04:03:10Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-21T13:00:03Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6103009
characteristics.characteristic: External Id
characteristics.text: UK Dementia Research Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2020-03-01T04:03:10Z
characteristics.characteristic: INSDC first public
characteristics.text: 2019-10-21T13:00:03Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3903692
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-8456:Input P525L 3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: HEK293T
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001184
characteristics.characteristic: cell line
characteristics.text: epithelial cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000066
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: HEK293T cells expressing wild type or P525L eGFP-tagged FUS fusion protein were lysed and subsequently subjected either to a co-IP experiment using anti-GFP nanobodies coupled to magnetic beads or to eGFP-FUS droplet purification. For co-IP, the lysates were subjected to GFP nanobodies coupled to magnetic beads after immunoprecipitation and subsequent washes, 1 ml TRIZOL was added directly to the beads for RNA isolation. Droplets were generated through reducing the volume of the lysate and stabilized using the reversible crosslinker formaldehyde. Thereafter, the droplets were purified by fluorescence activated particle sorting and additional wash steps and eluted in RNA-sample buffer (50 mM TRIS pH7.0, 5 mM EDTA, 1 % SDS, 10 mM DTT). To isolate RNA from the droplets, the formaldehyde crosslink was reversed through incubation of the droplets in RNA-sample buffer (50 mM TRIS pH7.0, 5 mM EDTA, 1 % SDS, 10 mM DTT) for 40 min at 70 °C. After cooling of the sample on ice for 5 min, 1 ml TRIZOL was added. HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum (FCS), penicillin (100 U/µl) and streptomycin (100 µg/ml)) at 37 °C and 5 % CO2. RNA was isolated from TRIZOL according to the manufacturer’s instructions Quality and quality of RNA was analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA isolated from transiently transfected cells served as input RNA. Total RNA isolated from droplets, from the co-IP and input RNA (in biological triplicates for each sample) were ribodepleted using the RiboMinus™ Transcriptome Isolation Kit (Invitrogen, K155004) before library preparation according to the manufacturer’s manual. Libraries were prepared using the strand-specific Illumina TruSeq Stranded Total RNA kit (Part # 15031048 Rev. E).
characteristics.characteristic: description
characteristics.text: embryo
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000922
characteristics.characteristic: developmental stage
characteristics.text: GFP-FUS_P525L
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0002113
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Input P525L 3
characteristics.characteristic: title