organism: Macaca nemestrina
description: Protocols: 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 the cell suspension, 50,000 cells were used as input for bulk ATAC-seq (as described in Buenrostro et al. 2013). All stem cell lines were 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. Human cerebral organoids were generated independently for the H9 embryonic stem cell line (from WiCell), and the induced pluripotent stem cell lines 409B2 (from the RIKEN BRC cell bank) and SC102A-1 (from System Biosciences). Chimpanzee cerebral organoids were generated independently for the induced pluripotent stem cell lines SandraA (a 7 year old female chimpanzee from Leipzig Zoo, Germany, when lymphocytes were obtained from her blood, then reprogrammed using plasmid based reprogramming per Okita et al. 2013; non-transgenic cell line), JoC (an 18 year old male chimpanzee from the Tchimpounga Sanctuary when lymphocytes were obtained from his blood, then reprogrammed using plasmid based reprogramming per Okita et al. 2013; non-transgenic cell line) and PR818-5 (originally generated by the Gage lab and kindly provided to us by the R. Livesey group). Bonobo cerebral organoids were generated for the induced pluripotent stem cell line Bokela (an 8 year old female bonobo from Leipzig Zoo, Germany, when fibroblasts were sampled from her skin, then reprogrammed using the StemMACS mRNA transfection kit; non-transgenic cell line). Macaque cerebral organoids were generated for the MN-1 embryonic stem cell line (kindly provided to us by the R. Livesey group who received them from Eliza Curnow). Following the protocol of Buenrostro et al. 2013, 50,000 cells were spun down in a refrigerated centrifuge (4°C) for 5 min at 500xg. The supernatant was removed and cells were washed with 50 uL cold PBS buffer, then spun down for 5 min at 500xg. The supernatant was removed and the cell pellet was resuspended in 50 uL of cold lysis buffer, then centrifuged for 10 min at 500xg. The supernatant was removed, the nuclei pellet was placed on ice, and we immediately proceeded to the transposition reaction. The nuclei pellet was resuspended in 50 uL cold transposition reaction mix (2.5 uL TDE1/Tn5 transposase from the Illumina Nextera kit, 25 uL TD 2x reaction buffer from the Illumina Nextera kit, 22.5 uL nuclease-free water) and incubated at 37°C for 30 minutes in a Thermomixer. The transposed DNA was purified using a QiaGen MinElute PCR Purification Kit. To determine the number of amplification cycles needed, we performed a qPCR side reaction using 100x SYBR Green 1. Transposed DNA from each sample was then amplified 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: 2019-08-07T14:39:55Z
attributes.tag: External Id
attributes.value: SAMEA5857966
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: 2019-08-07T14:39:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8228:Sample 25
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MN_1
attributes.tag: cell type
attributes.value: embryonic stem cell
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: sample name
attributes.value: E-MTAB-8228:Sample 25
attributes.tag: scientific_name
attributes.value: Macaca nemestrina
attributes.tag: sex
attributes.value: female