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Status |
Public on Sep 19, 2021 |
Title |
ciPS_C6_d15_batch1 |
Sample type |
SRA |
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Source name |
ciPS_C6
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Organism |
Pan troglodytes |
Characteristics |
cell type: chimpanzee neural forebrain progenitor cells chip antibody: n/a days in culture: 15 molecule subtype: polyA RNA
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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 |
polyA RNA |
Extraction protocol |
On the day of harvest, the cells were washed once with PBS and lysed with 350 µl RLT buffer with 1% mercaptoethanol (Thermo Fisher). RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturer’s protocol. The quality and concentration of the RNA was analyzed using 2100 Bioanalyzer (RNA nano; Agilent) and Qubit (RNA HS assay kit). TruSeq RNA Library Prep kit v2 (Illumina)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
iPS-derived chimpanzee neural forebrain progenitors RNAseq_H6_C6_map_hg38_pt6.tab 262
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Data processing |
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 |
SAMN20818932 |
SRA |
SRX11787879 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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