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| Status |
Public on May 30, 2022 |
| Title |
F2_Glu_45min_rep2 [s23] |
| Sample type |
SRA |
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| Source name |
NSB3188 (iPSC)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NSB3188 (iPSC)
cell type: induced glutamatergic neurons
treatment: depolarization
timepoint: 45min
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| Treatment protocol |
Prior to depolarization, medium was replaced with serum free Neurobasal A/B27/Glutamax for 3 days. The neuronal culture was then silenced using 1μM of Tetrodotoxin citrate (TTX) (Labome 1069/1) and 100μM of DL-2-amino-5-phosphopentnoic acid (DL-AP5) (Abcam ab120271) for 12-16h. The neurons were then depolarized by adding depolarization buffer (170 mM KCl, 2 mM CaCl2, 1 mM MgCl and 10 mM HEPES directly into the media to a final concentration of 30% of the total culture media. Neurons were collected at different time points after depolarization for experiments.
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| Growth protocol |
hES or hiPS cell–derived neurons were routinely generated by overexpression of the transcription factors Ngn2 (Glu neurons) or Ascl1 and Dlx2 (GABA neurons). On day 1, medium was replaced by N2 media(1x N2 supplement, 1X NEAA in DMEM-F12 media) containing 2 mg/ml Doxycycline to induce TetO expression. Selection of transduced cells was achieved with N2 media containing antibiotics for 2 or 3 days, and media was replaced with N2 media containing 4 μM Cytosine β-D-arabinofuranoside hydrochloride for 2 or 3 days to inhibit cell proliferation. Induced neurons were dissociated using Accutase and replated with mouse glial cells on Geltrex-coated plates. The media used for plating was a 1:1 mix of Neurobasal medium supplemented with B27, Glutamax containing 1% FBS and glial plating media(1x Glutamax , 2mg/ml Glucose and 5% FBS) containing 2 μM Thiazovivin(TV) and 2 mg/ml Doxycycline. TV was removed from media the following day. Half of the media was changed every 2-3 days and replaced with fresh Neurobasal medium supplemented with B27, Glutamax containing 1% FBS. 7 days later, the media was replaced by Neurobasal A, B27, Glutamax containing 1% FBS and half of it was replaced every 4 days. Mature neurons were co-cultured with mouse glial cells on day in vitro 35 (DIV35) for various experiments.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
DIV35 neurons were washed once with D-PBS, lifted using Accutase and RNA was extracted with TRIzol following the provider’s instructions. Following isolation, the RNA was treated with TURBO DNA-free kit. Total RNA (1μg) was used per sample for library preparation and sequencing.
Standard RNA-seq with Poly(A) mRNA selection was performed by Genewiz using Illumina NovaSeq platform and yielding 20-30 million paired-end 150 bp reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
F2 Ngn2 3/8 45'
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| Data processing |
Raw reads were trimmed for base call quality (PHRED score ≥ 21) using skewer 0.2.2 (Jiang et al., 2014).
Transcript quantification was performed using combined human-mouse reference transcriptome (GENCODE v28 and vM18) and salmon 1.13.1 (Patro et al., 2017), and human reference-specific counts extrated for downstream analysis..
Count normalization and FPKM calculation were performed using DESeq2 in R.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample
Supplementary_files_format_and_content: Salmon Counts (quant.sf) transcript abundance for every gene and every sample for both mouse and human transcriptomes
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| Submission date |
Feb 16, 2022 |
| Last update date |
May 30, 2022 |
| Contact name |
Nan Yan |
| E-mail(s) |
linda.lee2@mssm.edu, nan.yang1@mssm.edu
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| Organization name |
Mount Sinai
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| Department |
Neuroscience
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| Lab |
Yang Lab
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| Street address |
1425 Madison Ave
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE196855 |
Mapping cis-regulatory elements in human excitatory and inhibitory neurons links psychiatric disease heritability and activity-regulated transcriptional programs [RNA-seq] |
| GSE196856 |
Mapping cis-regulatory elements in human excitatory and inhibitory neurons links psychiatric disease heritability and activity-regulated transcriptional programs |
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| Relations |
| BioSample |
SAMN25995858 |
| SRA |
SRX14202872 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5903201_s23.quant.sf.gz |
1.9 Mb |
(ftp)(http) |
SF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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