organism: Macaca nemestrina
description: Protocols: The MN-1 macaque cell line is an embryonic stem cell line, so it is neither reprorgrammed or transgenic. We received this line from the Livesey group (University of Cambridge) who recieved them from Eliza Curnow (Washington National Primate Research Centre). The Livesey group used the MN-1 cell line in the paper Otani et al. 2016 (https://www.cell.com/cell-stem-cell/comments/S1934-5909(16)00109-0). Cells were cultivated using standard feeder-free conditions in StemMACS iPS-Brew XF (Myltenyi Biotec) on matrigel-coated plates and differentiated into cerebral organoids using a whole organoid differentiation protocol (Lancaster et al. 2014).For organoid experiments (2 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: 2019-07-08T12:08:58Z
attributes.tag: External Id
attributes.value: SAMEA5762177
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: 2019-07-08T12:08:58Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8087:Sample 232
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MN_1
attributes.tag: common name
attributes.value: pig-tailed macaque
attributes.tag: developmental stage
attributes.value: embryo
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: inner cell mass
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-8087:Sample 232
attributes.tag: scientific_name
attributes.value: Macaca nemestrina
attributes.tag: single cell well quality
attributes.value: OK