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| Status |
Public on Mar 20, 2024 |
| Title |
Ngn2 iNeurons REST WBS isoWBS REST inhibitor X5050 3 |
| Sample type |
SRA |
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| Source name |
isoWBS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: isoWBS
cell type: Ngn2 iNeurons
genotype: WBS
model: Isogenic
group: REST
treatment: REST inhibitor X5050
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| Growth protocol |
In order to obtain cortical glutamatergic neurons (iNeurons), iPSCs were dissociated with Accutase (GIBCO, Thermo Fisher Scientific) and plated on Matrigel-coated plates (final 2% v/v, Corning) in mTeSR™ or TeSR-E8 (Stemcell Technologies) supplemented with 5 μM Y-27632 (Sigma). iPSCs were then cultured in MEM1 (DMEM/F12 1:1 (Euroclone/Gibco) supplemented with NEAA 1%, N2 1%, BDNF 10 ng/ml, NT-3 10 ng/ml, Laminin 0,2 μg/ml and 2 μg/ml doxycycline hydrochloride, penicillin 100 U/ml and streptomycin 100 μg/ml) for 2 days. On the second day of MEM1, cells were selected with 1 μg/ml puromycin, to reassure that only the cells with Ngn2-inducible cassette will survive. For patient-derived iNeurons, after two days of MEM1, media was change to Neurobasal medium (NB, Thermo Fisher Scientific) supplemented with BDNF 10 ng/ml, NT-3 10 ng/ml, B27 (1:50), GlutaMax (Gibco, 1:100), penicillin 100 U/ml, streptomycin (100 μg/ml), which was previously conditioned on mouse astrocytes for 24 h. Half of media was changed every other day. Instead, for isogenic iNeurons after two days of MEM1, media was change to Neurobasal Plus medium (NB-Plus) composed of B-27™ Plus Neuronal Culture System (Thermo Fisher Scientific) supplemented with Glutamax 0.25% (Thermo Fisher Scientific), 2 μg/ml, doxycycline hydrochloride and penicillin 100 U/ml, streptomycin (100 μg/ml). The media was changed twice a week. On day 7-8, cells that already acquired neuron-like shape were dissociated with Accutase, counted and seeded into plates coated with 15 μg/ml of poly-D-lysine at a density of 1 x 10^6 cells/well of multiwell 6, 2 x10^6 in 6 cm dishes or 4 x10^6 in 10 cm dishes (Nunc Edge plates, Thermo Fisher Scientific) in conditioned NB or NB-Plus. Half of media was changed twice a week until day 30-35.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated using the RNeasy mini Kit (QIAGEN) and genomic DNA was removed using the RNase-Free DNase Set (QIAGEN). Retrotranscribed cDNA was obtained from 1 μg of total RNA using the SuperScript VILO Retrotranscription kit from Life Technologies according to the manufacturer’s instructions.
Library preparation for RNA-seq was done with the Ribo-Zero Total RNA sample preparation kit (Illumina), starting from 250 ng to 1 g of total RNA. The quality of cDNA libraries was assessed by Agilent 2100 Bioanalyzer using the High Sensitivity DNA Kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RNA.restExperiment.SE.rds
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| Data processing |
Quantification was done using Salmon 0.9.1 (Patro et al., 2017) on the ensembl 92 transcriptome.
Only protein-coding transcripts were retained, and counts were aggregated at the level of gene symbols.
Only genes with at least 20 reads in at least a number of samples corresponding to 75% of the smallest experimental group were included in the analysis.
Differential expression analysis was done using DESeq2 (Love et al., 2014), and for pairwise comparisons between groups foldchanges were shrunken using the apeglm method (Zhu et al., 2019). In addition, a regression on 7q11.23 copy-numbers was performed.
Assembly: GRCh38
Supplementary files format and content: RNA.SE.rds.zip are RNA.restExperiment.SE.rds.zip are R SummarizedExperiment objects containing the quantification and annotation for the different samples.
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| Submission date |
Mar 15, 2024 |
| Last update date |
Mar 20, 2024 |
| Contact name |
Alessandro Vitriolo |
| E-mail(s) |
alessandro.vitriolo@fht.org
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| Organization name |
Fondazione Human Technopole
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| Department |
Neurogenomics
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| Street address |
Via Rita Levi Montalcini 1
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| City |
Milano |
| State/province |
MI |
| ZIP/Postal code |
20157 |
| Country |
Italy |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE261691 |
7q11.23 CNV alters ribosomal biogenesis, mTOR and neuronal intrinsic excitability [RNA-seq] |
| GSE261692 |
7q11.23 CNV alters ribosomal biogenesis, mTOR and neuronal intrinsic excitability |
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| Relations |
| BioSample |
SAMN40469270 |
| SRA |
SRX23958574 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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