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Status |
Public on Jul 13, 2016 |
Title |
LVNC sibling1 hiPSC-CMs at 2 weeks #1 |
Sample type |
SRA |
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Source name |
cardiomyocytes
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Organism |
Homo sapiens |
Characteristics |
cell type: induced pluripotent stem cell derived cardiomyocytes passages: 25-30 genotype: TBX20 wt/Y317* cardiac phenotype: asymptomatic LVNC
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Treatment protocol |
hiPSC-CMs were cultured in RPMI with B27 supplement and 2% FBS 2 days before experiment to accelerate the proliferation.
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Growth protocol |
hiPSCs were grown to 90% confluence and differentiated subsequently into beating cardiomyocytes using a small molecule-based monolayer method adapted after4 and described in detail previously.3 10 days after cardiac differentiation, hiPSC-CM monolayers were purified using non-glucose culture medium containing RPMI-1640 without glucose (Life technologies) and B27 supplement (Life technologies). Non-glucose culture medium was changed every two days and after 5 days, hiPSC-CMs were dissociated using TripleE (10 min), washed and plated onto matrigel-coated plates in culture medium with glucose.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Total RNA was extracted and quantified using miRNeasy Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol. An additional DNAseI digestion step was performed to ensure that the samples were not contaminated with genomic DNA. Following the Qiagen RNA sample preparation protocol, 10 ug of total RNA was used to generate index-tagged paired-end cDNA libraries.5 Briefly, mRNAs were purified by polyA enrichment procedure using Dynal Oligo(dT) beads (Life technologies). mRNA fragmentation was performed using RNA Fragmentation Reagents (Life technologies) with incubation at 70oC for 1 min to obtain 200-300b fragments. cDNA was generated using SuperScript Double-Stranded cDNA Synthesis Kit (Life technologies). Illumina sequencing adapters were ligated to cDNA using LigaFast (Promega) and PE Adapter Oligo Mix (Illumina, San Diego, CA). PCR was performed on the adapter-ligated cDNA with 2X Phusion DNA polymerase Master Mix (New England Biolabs, Ipswich, MA) using the following conditions: denaturation 98°C for 30 seconds, followed by 15 cycles of denaturation 98°C for 10 seconds, annealing 65°C for 30 seconds, and extension 72°C for 30 seconds, ending with an extension at 72°C for 5 min. After cDNA libraries were generated using the sample preparation kit, quality of the libraries was checked using Bioanalyzer (Agilent Technologies).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
Paired-end fastq sequence reads from each sample were assembled against hg19 using the Tophat v2.0.6 with the Illumina-supplied hg19 gene-model annotation file ( gtf annotation). The expression level (FPKM) was estimated by Cufflinks. Cuffdiff was used to call differentially expressed genes with false discovery rate less than 0.05. Cuffdiff was run against the UCSC iGenomes GTF file from Illumina. Subsequent to determining which genes were differentially expressed, FPKM was used for filtering, and visualization purposes. Genome_build: hg19
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Submission date |
Nov 10, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Kazuki Kodo |
E-mail(s) |
kkodo@stanford.edu
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Organization name |
Stanford
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Street address |
campus street
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City |
stanford |
ZIP/Postal code |
94305 |
Country |
USA |
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Platform ID |
GPL11154 |
Series (1) |
GSE63161 |
hiPSCs unravel aberrant TGFβ signaling as an etiology of left ventricular non-compaction |
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Relations |
BioSample |
SAMN03174785 |
SRA |
SRX757102 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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