title: Pooled_set2_seg1_2
organism: mixed sample
description: Protocols: For nuclei isolation from frozen tissue, all following steps were performed on ice with precooled buffers and centrifugation steps were performed at 4°C. Briefly, tissue was spun down, thawed on ice and 1 ml PBSE (PBS (Gibco), 2 mM EDTA (Life Technologies)) was added to the tissue. The tissue slices were incubated at 4°C on a shaker at 1500 rpm for a total of 45-60 min with trituration steps in between using 1000p and 200p to homogenize the tissue. Generally, segments 1-10 were used for single-nucleus experiments. Two segments were pooled to obtain sufficient material for single nucleus isolation, resulting in 5 segments per individual. To reduce batch effects and increase the number of nuclei per experiment, material from three different individuals (originating from human, chimp/bonobo and macaque respectively) was pooled for each segment. After homogenization, solutions were combined in a 5 ml tube and spun down at 900xg for 5 min. The pellet was resuspended in 1.5 ml PBSE + 1% NP-40 (BioVision), triturated 20 times using 1000p and incubated for 7 min incubation on ice. Samples were then spun down at 900xg for 5 min and resuspend in 1.5 ml PBSE + 1% BSA (Serva) two times. Samples were then spun down again at 900xg for 5 min and resuspended in PBS + 1% BSA. Before sorting, samples were filtered through 30 um cell filter (Miltenyi Biotec) and stained using DAPI (1:1000, BD Pharmingen). Nuclei were sorted in yield sort mode (BD FACS AriaIII and BD FACS Fusion) based on a defined nuclei population by excluding debris using FSC and SSC and by sorting DAPI positive events. Nuclei were sorted in bulk into 96 well plates and spun down 5 min at 600xg to enrich for nuclei in the pellet. Cubes were dissected from prefrontal cortex from human, chimpanzee, bonobo and macaque on dry ice aiming for cubes with few curvature to obtain reproducible slicing results. Briefly, the thickness of grey matter at all facets of the cube was measured to obtain a mean gray matter thickness. The mean thickness was divided by 10 to obtain the thickness for each of the segments, whereby each of the segments consisted of several slices at 50 um thickness. Sectioning was performed in a cryostat (Microm, Thermo Fisher). Single stranded cDNA was cleaned up and amplified after breaking the droplets generated during the sample collection using the 10X 3' Single cell Kit v2 according to the manufacturer's instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay. Libraries were constructed following the 10X library construction protocol v2. Briefly, cDNA was fragmented, end repaired and adaptor sequences were ligated, followed by a PCR to introduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-04T04:04:47Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-08-08T20:11:06Z
attributes.tag: External Id
attributes.value: SAMEA5858513
attributes.tag: INSDC center name
attributes.value: Department of Biosystems Science and Engineering, ETH Zurich
attributes.tag: INSDC first public
attributes.value: 2019-10-04T04:04:47Z
attributes.tag: INSDC last update
attributes.value: 2019-08-08T20:11:06Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8230:Pooled_set2_seg1_2
attributes.tag: age
attributes.value: 54/45/7
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: HG1/CHD2/MG2
attributes.tag: organism part
attributes.value: prefrontal cortex
attributes.tag: sample name
attributes.value: E-MTAB-8230:Pooled_set2_seg1_2
attributes.tag: scientific_name
attributes.value: mixed sample
attributes.tag: sex
attributes.value: male, female, and male
attributes.tag: species mix
attributes.value: Homo sapiens, Pan troglodytes, Macaca mulatta