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| Status |
Public on Sep 04, 2024 |
| Title |
ZIC3 WT, long-read, biol rep 2 of 3 |
| Sample type |
SRA |
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| Source name |
H1-OCT4-eGFP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H1-OCT4-eGFP
cell type: human embryonic stem cell
genotype: ZIC3 wildtype (ZIC3 WT)
treatment: Undifferentiated - cultured in mTeSR1 media supplemented with 100 µg/mL of geneticin
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| Treatment protocol |
Undifferentiated - cultured in mTeSR1 media supplemented with 100 µg/mL of geneticin. Three short-read RNA-seq samples also received 100 µg/mL cyloheximide (CHX) for 6 hr prior to RNA extraction.
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| Growth protocol |
Wildtype, ZIC3 c.1224+3286A>G, and ZIC3 knockout (KO) H1-OCT4-EGFP cells were cultured following the WiCell Feeder Independent Stem Cell Protocols using geneticin (G418 sulfate; Gibco) standard operating procedure 208 version 2.0. Briefly, 6-well plates (Corning) were coated with Matrigel® Matrix (growth factor reduced) diluted in DMEM/F-12, HEPES media (Invitrogen). After thawing, cell lines were cultured in Matrigel®-coated 6-well plates with mTeSR1 media (STEMCELL Technologies Inc.). Cells were maintained in mTeSR1 media for an additional 24 h, then replaced with mTeSR1 media supplemented with 100 µg/mL of geneticin (mTeSR1-G). Cells were dissociated using Versene solution (Gibco) and collected for further experiments.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) followed by column purification with the RNeasy MinElute Cleanup kit (Qiagen).
Short-read RNA-seq was completed by the Indiana University School of Medicine Center for Molecular Genomics. Total RNA was extracted from wildtype, ZIC3 AtoG_C1, ZIC3 AtoG_C2, ZIC3 KO_C1, and ZIC3 KO_C2 H1-OCT4-EGFP cells. The RNA integrity number (RIN) was determined using an Agilent TapeStation (Agilent Technologies) followed by an RNA purification step with the KAPA RNA HyperPrep kit (Roche). cDNA libraries were prepared using the NovaSeq 6000 SP Reagent kit v1.5 (300 cycles) and sequenced using the NovaSeq 6000 (Illumina) with 2 x 150 bp paired-end reads. Long-read RNA-seq was performed by the Genome Access Technology Center at Washington University in St. Louis. Total RNA was extracted from control (ZIC3 WT) (n=3), ZIC3 AtoG_C1 (n=3), and ZIC3 KO_C1 (n=3) H1-OCT4-EGFP cells and the RIN determined with an Agilent Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared with the PCR-cDNA Barcoding kit SQK-PCB111.24 (Oxford Nanopore Technologies) and the Agilent Bioanalyzer (Agilent Technologies) served for quality assessment. Cell samples were pooled together on three PromethION R9.4.1 flow cells (50 fM/flow cell) and sequenced in a PromethION 24 A100 (Oxford Nanopore Technologies) for 90 h.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
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| Description |
ZIC3_WT_ONT_Flowcell_BiolRep2
ZIC3_WT_2
Long_Read_Log_CPM_EdgeR.txt
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| Data processing |
For the short-read RNA-seq, results were aligned to the GRCh38/hg38 reference genome using two passes of the Spliced Transcripts Alignment to a Reference (STAR) aligner, version 2.7.10b and visualized in the Integrated Genomics Viewer (IGV).
For the long-read RNA-seq, the following software was used: MinKNOW v22.12.5, Bream v7.4.8, Configuration v5.4.7, Guppy v6.4.6, MinKNOW Core v5.4.3. Bases were called using Guppy v6.4.6 running the super-accurate basecalling model, 450 bp setting. The fastq files from each of the three flow cells were merged together based on the sequencing barcode. Results were aligned to the GRCh38/hg38 reference genome using minimap2 and visualized in IGV. Differential expression analysis was conducted using the R package edgeR (version 4.0.2) comparing undifferentiated ZIC3 WT, ZIC3 AtoG_C1, and ZIC3 KO_C1 H1-OCT4-eGFP cells.
Assembly: GRCh38/hg38
Supplementary files format and content: tab-deliminated text file includes raw counts for all short-read RNA-seq samples - Short_Read_Raw_Counts_All_Samples.txt
Supplementary files format and content: tab-deliminated text file includes log_CPM values for all long-read RNA-seq samples - Long_Read_Log_CPM_EdgeR.txt
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| Submission date |
Apr 08, 2024 |
| Last update date |
Sep 04, 2024 |
| Contact name |
John Robert Wells |
| E-mail(s) |
[email protected]
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| Organization name |
Indiana University School of Medicine
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| Department |
Department of Medical & Molecular Genetics
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| Lab |
Stephanie Ware Lab
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| Street address |
1044 W Walnut St
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| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE263414 |
Non-coding cause of congenital heart defects: Abnormal RNA splicing with multiple isoforms as a mechanism for heterotaxy |
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| Relations |
| BioSample |
SAMN40889548 |
| SRA |
SRX24193963 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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