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| Status |
Public on Sep 19, 2021 |
| Title |
CR017_S7 |
| Sample type |
SRA |
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| Source name |
cPT5-H3K9me3
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| Organism |
Pan troglodytes |
| Characteristics |
cell type: chimpanzee neural forebrain progenitor cells
chip antibody: H3K9me3
days in culture: 14
molecule subtype: DNA released by antibody-targeted MNase
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| Treatment protocol |
Differentiation to fbNPCs: iPSCs were grown to approximately 70-90% confluency and were then dissociated as usual for passaging. After centrifugation, the cells were resuspended in N2 medium (1:1 DMEM/F-12 (21331-020; Gibco) and Neurobasal (21103-049; Gibco) supplemented with 1% N2 (Gibco), 2 mM L-glutamine (Gibco), and 0.2% penicillin/streptomycin). The cells were manually counted twice and plated at a density of 10,000 cells/cm2 in 250 µl medium/cm2 on LN111 Nunc multidishes (1.14 µg/cm2; Biolamina). 10 µM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) for dual SMAD inhibition, and 10 µM Y27632 was added to the medium. The medium was changed every 2-3 days (N2 medium with SB431542 and noggin) until day 9 of differentiation, when N2 medium without SMAD inhibitors was used. On day 11, the cells were replated by washing twice with DPBS followed by adding StemPro accutase (75 µl/cm2; Gibco) for 10-20 minutes at 37 ºC. The dissociated cells were washed off with 10 ml wash medium, centrifuged for 5 minutes at 400g and resuspended in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (Gibco), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 µM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma). The cells were counted and replated at 800,000 cells/cm2 on LN111- coated plastic in B27 medium (600 µl medium/cm2). The cells were kept in the same medium until day 14, after which new B27 medium was added (Grassi et al., 2020).
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| Growth protocol |
We used two human iPSC lines generated by mRNA transfection (RBRC-HPS0328 606A1 and RBRC-HPS0360 648A1, both from RIKEN; from here on referred to as HS1 and HS2, respectively). We used two chimpanzee iPSC lines: one generated by mRNA transfection (Sandra A, herein referred to as PT1) and the other with viral vector transduction (PR00818 PTCL-5, herein referred to as PT2) (Marchetto et al., 2013; Mora-Bermúdez et al., 2016). iPSCs were maintained on LN521-coated (0.7 µg/cm2; Biolamina) Nunc multidishes in iPS media (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (Gibco)). Cells were passaged 1:2-1:6 every 2-4 days by being rinsed once with DPBS (Gibco) and dissociated using 0.5 mM EDTA (75 µl/cm2; Gibco) at 37 ºC for 7 minutes. Following incubation, EDTA was carefully aspirated from the well and the cells were washed off from the dish using washing medium (9.5 ml DMEM/F-12 (31330-038; Gibco) and 0.5 ml knockout serum replacement (Gibco)). The cells were then centrifuged at 400g for 5 minutes and resuspended in iPS brew medium supplemented with 10 µM Y27632 (Rock inhibitor; Miltenyi) for expansion.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
On the day of harvest, the cells were washed once with PBS, detached using Accutase, washed and resuspended in PBS. Briefly, 2x105 cells were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche complete protease inhibitors) and attached to pre-activated ConA-coated magnetic beads (Bangs Laboratories). Bead-bound cells were resuspended in 50 µL buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 2 mM EDTA) containing primary antibody (H3K9me3 (Ab8898, Abcam 1:50); H3k4me3 (Cat #39159, Active Motif 1;50) or IgG control antibody (Ab97047, Abcam 1:50) at 4 ºC over-night. Washed with digitonin buffer, pA-MNase was added and incubated with the cells at 4 ºC for 1 h. Bead-bound cells were washed, resuspended in 100 µL digitonin buffer, and chilled to 0-2 ºC. Genome cleavage was stimulated by addition of 2 mM CaCl2 at 0 ºC for 30 min. The reaction was quenched by addition of 100 µL 2x stop buffer. After 10 min incubation at 37 ºC to release genomic fragments, cells and beads were pelleted by centrifugation (16,000 g, 5 min, 4 ºC) and fragments from the supernatant purified.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
iPS-derived chimpanzee neural forebrain progenitors
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| Data processing |
Library strategy: CUT&RUN
RNA-seq: raw base call files were converted to fastq and demultiplexed using bcl2fastq. The fastq files were mapped to hg38 and pantro6 reference genomes using STAR/2.5.0a with parameter --outFilterMismatchNoverLmax 0.03 and --sjdbGTFfile gencode.v27.annotation.gtf. When mapping to pantro6, the gencode.v27.annotation.gtf were lifted from hg38 coordinates to pantro6. Gene counts were quantified using featurecounts from Subread/1.5.0-p1, with -s 2, -p and -t exon parameters.
single-cell RNA-seq: raw basecall files were converted to fastq and demultiplexed using cellranger/2.1 mkfastq. Gene counting was done with cellranger count.
Genome_build: hg38
Supplementary_files_format_and_content: counts and coverage tracks
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| Submission date |
Aug 16, 2021 |
| Last update date |
Sep 19, 2021 |
| Contact name |
Johan Jakobsson |
| E-mail(s) |
johan.jakobsson@med.lu.se
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| Organization name |
Lund University
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| Department |
Wallenberg Neuroscience Center
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| Lab |
Molecular Neurogenetics
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| Street address |
Sölvegatan 17
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| City |
Lund |
| ZIP/Postal code |
22362 |
| Country |
Sweden |
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| Platform ID |
GPL21121 |
| Series (1) |
| GSE182224 |
Structural variation at the ZNF558 locus controls a gene regulatory network in human brain development |
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| Relations |
| BioSample |
SAMN20818952 |
| SRA |
SRX11787894 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5525344_cPT5-H3K9me3-CR017-hg38-scaled.bw |
99.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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