organism: Pan paniscus
description: Protocols: Bokela was an 8 year old female bonobo from Leipzig Zoo (Germany) when fibroblasts were sampled from her skin. Fibroblasts were reprogrammed using the StemMACS mRNA transfection kit (Miltenyi Biotec). The cell line was validated for pluripotency markers by immunhohistochemical stainings using the Human Pluripotent Stem Cell 3-Color Immunohistochemistry Kit (R&D Systems, SC021) and was differentiated into the three different germ layers using the Human Pluripotent Stem Cell Functional Identification kit (R&D Systems) and StemMACS Trilineage Differentiation Kit (Miltenyi Biotec). Karyotyping was carried out using Giemsa banding at the Technische Universitaet Dresden Stem Cell Engineering facility and karyotypes were found to be normal. The cell line is not transgenic. The Bokela iPSC line was cultured using standard feeder-free conditions in mTeSR1 (StemCell Technologies) on matrigel-coated plates and differentiated into cerebral organoids using a whole organoid differentiation protocol as described in Lancaster et al. 2014. For organoid experiments (2 months, 4 months), organoids were washed twice with PBS in a Tissue-Tek Cryomold (Sakura), then embedded in 4% low-melting agarose (Sigma) and sliced into 150 μm sections using a vibrating microtome (Ci 7000 smz, Camden Instruments). Slices were placed on microscope slides containing differentiation plus vitamin A (Diff +VA) media and inspected under a stereomicroscope (Leica) to dissect cortical regions. Selected regions were washed twice in 500 μL PBS and incubated at 37°C in 500 μL Accutase (Sigma) plus 0.5 μL DNase I (New England Biolabs) for ~45 minutes. Trituration was performed for additional mechanical dissociation. Cells were passed through a 30 μm pre-separation filter (Miltenyi Biotec), washed with Diff+VA medium, and spun down at 300 x g (Heraeus Megafuge 40R, Thermo Scientific) for 5 minutes. The cell pellet was resuspended in 200 μl of Diff+VA medium. Cells were viewed under a microscope (Zeiss) to ensure a single cell suspension was obtained, and then counted using a Countess Automated Cell Counter (Invitrogen). From this cell suspension, cells were diluted to a final concentration of 300 cells/μl and used for microfluidics based single-cell ATAC-seq. Cells were mixed with Suspension Reagent (Fludigm) at a 3:2 ratio and loaded onto a primed medium (10-17 μm) integrated microfluidic circuit (Fludigm) for capturing. Lysis, transposition, and amplification were performed on the Fluidigm C1 platform. DNA from each cell was transferred to an individual well of a 96-well plate and barcoded with unique combinations of 24 adapter-index i7 and 16 adapter-index i5 primers. Quantification and library size distribution was assessed on an Agilent 2100 Bioanalyzer using High Sensitivity DNA chips. Excessive primer contamination was removed using SPRIselect (Beckman Coulter Life Sciences) size selection.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-16T17:04:26Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-07-03T11:00:54Z
attributes.tag: External Id
attributes.value: SAMEA5752622
attributes.tag: INSDC center name
attributes.value: MPI-EVA
attributes.tag: INSDC first public
attributes.value: 2019-10-16T17:04:26Z
attributes.tag: INSDC last update
attributes.value: 2019-07-03T11:00:54Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8083:Sample 180
attributes.tag: age
attributes.value: 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: induced pluripotent stem cell
attributes.tag: common name
attributes.value: pygmy chimpanzee
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: organoid culture
attributes.tag: organism part
attributes.value: skin
attributes.tag: progenitor cell type
attributes.value: skin fibroblast
attributes.tag: sample name
attributes.value: E-MTAB-8083:Sample 180
attributes.tag: scientific_name
attributes.value: Pan paniscus
attributes.tag: sex
attributes.value: female
attributes.tag: single cell well quality
attributes.value: OK