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| Status |
Public on Jun 25, 2015 |
| Title |
D3_meso_3_7 |
| Sample type |
SRA |
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| Source name |
RUES2 human embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
differentiation stage: mesoderm
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| Treatment protocol |
The differentiation set up was initiated by plating undifferentiated hESCs as single cells. The cultures were treated with CHIR-99021 (Cayman chemical,13122) for 24 hours before reaching confluence. Cells were induced to differentiate (designated day 0) by replacing the culturing media with RPMI media (Invitrogen, 11875-119) containing 100 ng/mL Activin A (R&D SYSTEMS, 338-AC-050), 1:60 diluted Matrigel (BD), and insulin-free B27 supplement (Invitrogen, 0050129SA). An RPMI media change the following day (17 hours) included different 5 ng/mL BMP4 (R&D SYSTEMS, 314-BP-050), 1 uM CHIR-99021, and insulin-free B27 supplement. On day 3 of differentiation media was changed to RPMI media containing 1 uM XAV-939 (Tocris, 3748) and insulin-free B27 supplement. RPMI containing insulin-free B27 supplement was utilized until differentiation day 7 in which the media is replaced with RPMI containing a B27 supplement that includes insulin (Invitrogen, 17504044). Subsequent media changes included the insulin-containing supplement.
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| Growth protocol |
RUES2 human embryonic stem cells were maintained in mouse embryonic fibroblast-conditioned media
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| Extracted molecule |
total RNA |
| Extraction protocol |
Between 1-3 million cells per sample were pelleted for each sample. RNA was extracted as per the manufacturer's protocol for the mirVana kit (Life Technologies). After extraction, RNA was quantified using the Quant-iT RNA BR Assay Kit (Life Technologies). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent) using RNA Nano-chips. Following RNA isolation (mirVana miRNA Isolation Kit, Life Technologies, Inc.), the RNA was quantified (Qubit RNA Assay Kit, Life Technologies, Inc.), and quality controlled (RNA6000 Nano Kit and BioAnalyzer 2100, Agilent)
200 ng to 1000 ngwas used as input for the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold sample prep kit (Illumina, Inc.) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, first both cytoplasmic and mitochondrial rRNA was removed by selectively hybridizing biotinylated probes to target sequences and using magnetic beads to capture the bound products. Following rRNA removal, the RNA was fragmented and copied into first strand cDNA using random primers and reverse transcriptase. Next, second strand cDNA synthesis was completed using DNA Polymerase I and RNase H. The cDNA was then ligated to Illumina supplied adapters and enriched with PCR to create the final cDNA libraries. The libraries were then pooled and sequenced on a HiSeq 2000 (Illumina, Inc.) instrument as per manufacturer’s instructions. Sequencing was performed up to 2 X 101 cycles.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw fastq files were processed using TrimGalore version 0.3.7 and cutAdapt version 1.5 to remove adapter sequences and trim poor-quality bases. All default values were used, with the exceptions that the length parameter was set to exclude reads where either mate had a trimmed length of less than 50
Circular and linear junction reads were called using custom scripts available with publication
Genome_build: hg19
Supplementary_files_format_and_content: txt files were generated by custom scripts available with publication; pvalue represents the posterior probability from the GLM method (high posterior probability indicates high confidence)
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| Submission date |
Dec 22, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Linda Szabo |
| Organization name |
Stanford University
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| Department |
Biochemistry
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| Lab |
Salzman Lab
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| Street address |
Beckman Center, B400 279 W. Campus Dr. MC: 5307
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE64417 |
Induction of circular RNA in fetal heart development recapitulated in in vitro differentiation |
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| Relations |
| BioSample |
SAMN03272103 |
| SRA |
SRX821436 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1571092_s_007_T21_mesoderm_4_ATGTCA_L006_R1_circJuncProbs.txt.gz |
14.0 Kb |
(ftp)(http) |
TXT |
| GSM1571092_s_007_T21_mesoderm_4_ATGTCA_L006_R1_linearJuncProbs.txt.gz |
2.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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