organism: Homo sapiens
description: Protocols: 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. All the protocols are the same as in E-MTAB-8083 (ie as described in Lancaster et al. 2014) Cerebral organoids were generated independently for the H9 embryonic stem cell line (from WiCell) and the 409B2 induced pluripotent stem cell line (from the RIKEN BRC cell bank). During the experiments, we took an equal number of H9 organoids and 409B2 organoids, dissociated the organoids into single cells independently for each line, and then obtained a cell count for each line. Finally, we took an equal number of H9 cells and 409B2 cells (1:1 ratio) and loaded this cell mixture into the Fluidigm IFC for capturing and performing the single-cell ATAC-seq protocol. Description and phenotype annotation is as follows: d30: this refers to organoids after 30 days differentiation but no clear cell type; EB: this refers to embryoid bodies prior to organoid differentiation; iPSC: this refers to induced pluripotent stem cells prior to embryoid body or organoid culture; NEcto: this refers to organoids where cells have differentiated to neuroectoderm fate; NEpith: this refers to organoids where cells have differentiated to neuroepithelial fate; organoid: this refers to organoids where no cell fate was determined 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: 2019-10-16T17:04:27Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-07-31T12:50:29Z
attributes.tag: External Id
attributes.value: SAMEA5820191
attributes.tag: INSDC center name
attributes.value: MPI-EVA
attributes.tag: INSDC first public
attributes.value: 2019-10-16T17:04:27Z
attributes.tag: INSDC last update
attributes.value: 2019-07-31T12:50:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8089:Sample 634
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 409B2
attributes.tag: cell type
attributes.value: induced pluripotent stem cell
attributes.tag: common name
attributes.value: human
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: fibroblast
attributes.tag: sample name
attributes.value: E-MTAB-8089:Sample 634
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: single cell well quality
attributes.value: OK