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| Status |
Public on Mar 12, 2021 |
| Title |
CTRL_iPSC_2 |
| Sample type |
SRA |
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| Source name |
iPSC
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| Organism |
Homo sapiens |
| Characteristics |
nrxn1-genotype: WT
sample group: ctl -iPSC
culture batch pair-group: iPSC
nrxn1 transcript mutation effect: none
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| Treatment protocol |
Neurons from engineered iPS cells were treated with the same protocol except additional treatment of lentivirus expressing Ubiquitin-Flp-GFP and Ubiquitin-Cre-GFP at Day -1.
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| Growth protocol |
Neurons were generated from feeder-dependent iPS cells: (Day -1) iPS cells were plated and infected on Matrigel coated 24-well plate. Per 6-well of iPS cells grown on feeders, cells were washed 1x with PBS. 0.5 ml of CTK (0.25% Trypsin no phenol red, 0.1 mg/ml collagenase IV, 0.5 ml 1 mM CaCl2, 20% KnockOut Serum Replacement in sterile water) was added to remove the feeders at 37°C until feeder cells lift off. CTK was removed and washed 2x with PBS. iPS cells were treated with 0.5 ml Accutase briefly, resuspended in 1 ml hES media and spun down at 150 g x 5 min. Cell pellets were resuspended in 50% hES media/50% mTeSR plus Y-27632, and lentiviruses. Typically 200,000 - 300,000 cells were plated per well of a 24-well plate with 1 uL of viruses (rtTA and Ngn2-puro) in 1 ml of mixed media. (Day 0) Half of the virus-containing media was removed and fresh Neural induction medium (NIM; DMEM F12 medium supplemented with 1% N2, 1% non-essential amino acids, 10 μg/l human BDNF, 10 μg/l human NT-3, and 0.2 mg/l mouse laminin) was added supplemented with Doxycycline (2 mg/l) and Y-27632 to induce Ngn2-puro expression. (Day 1-2) 24 hours later, Puromycin (1 mg/l) was added to NIM for 24-48 hours and media was changed daily. (Day 3) neurons were washed 1-2x with PBS. Freshly isolated or previously cryopreserved mouse glial cells (~75,000 cells per well of a 24-well plate) were added in Neural differentiation medium plus Dox (NDM; Neurobasal A medium supplemented with 2% B27, 1% glutamax, 2 μM AraC, 10 μg/l human BDNF, 10 μg/l human NT-3, and 0.2 mg/l mouse laminin). (Day 5) Half of the media was changed using NDM plus Dox. Media change occurred every 2-3 days until Day 10. (Day 10) Half of media was replaced with Maturation medium (MM; MEM supplemented with 0.5% glucose, 0.02% NaHCO3, 0.1 mg/ml transferrin, 5% FBS, 0.5 mM L-glutamine, 2% B27, and 2 μM AraC). Dox was withdrawn from this time point forward. (Day 13) Additional MM (0.5 mL per well of a 24-well plate) was added and neurons were incubated for 7 days without media change. (Day 20) Half of media was replaced with fresh MM. Media change occurred weekly until RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from the entire plate and thus contained both mouse and human total RNA
RNA-seq libraries were prepared from total RNA using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
none
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| Data processing |
Illumina Casava1.7 software used for basecalling. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence and converted into read1 and read2 fastq files from each sample.
RNA-seq reads were aligned to a combined human + mouse transcriptome reference (Ensembl Mus_musculus GRCm38 and Homo_sapiens. GRCh38 transcriptomes v94) using the stranded library option of Kallisto v0.46.0 (Bray et al., 2016) using a concatenated mouse-human index. Based on comparisons with pure mouse and human RNA samples, the Kallisto pseudo-alignment algorithm was able to accurately distinguish and assign transcripts to the proper species reference.
From each Kallisto-processed mouse-human RNAseq sample, an expression matrix was constructed to generate human-specific TPMs (transcripts per million) as follows. Human transcript counts and TPM values were extracted and adjusted based on (1) per million human reads per mouse-human sample; (2) transcript-level TPM values were summed per gene; (3) gene TPM values were converted to log2(TPM+1) values, (4) 19,701 genes were selected with log2(TPM+1) ≥ 1 in at least one of the thirty samples, and (5) the resulting gene-level values were per-sample quantile-polished to reduce sample-sample variability (Zyprych-Walczak et al., 2015). Genes for subsequent analyses were selected from 17,004 gene IDs with log2(TPM+1) ≥ 2 in at least one of the thirty samples. Below we refer to log2(TPM+1) values as “logTPM” values.
DEG analyses used “moderated” t-tests as implemented in limma v3.42.2 (Law et al., 2014) with the limma-trend procedure (fit = eBayes(fit, trend = TRUE)) that uses an Empirical Bayes Model to effect “shrinkage of the estimated sample variances towards a pooled estimate” (Smyth 2004) which can increase power in the face of culture-culture variability. The use of this approach on logTPM data is conservative, which we consider appropriate with a small cohort. R scripts for DEG analyses are included as supplementary material.
Genome_build: Ensembl Mus_musculus GRCm38 and Homo_sapiens. GRCh38 transcriptomes v94.
Supplementary_files_format_and_content: Kallisto output file
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| Submission date |
Mar 11, 2021 |
| Last update date |
Mar 12, 2021 |
| Contact name |
Bruce J Aronow |
| E-mail(s) |
bruce.aronow@cchmc.org
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| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
Biomedical Informatics, Developmental Biology
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| Lab |
Genome Informatics
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| Street address |
240 Albert Sabin Way
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE168762 |
Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons |
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| Relations |
| BioSample |
SAMN18270191 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5169064_3iPSC_abundance.tsv.gz |
953.5 Kb |
(ftp)(http) |
TSV |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data not provided for this record |
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