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| Status |
Public on May 31, 2021 |
| Title |
E24NocR1 |
| Sample type |
SRA |
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| Source name |
H9 cells
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| Organism |
Homo sapiens |
| Characteristics |
type: embryonic stem cells
treatment: Mesendoderm 24h + Nocodazole
biological replicate: Replicate 1
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| Treatment protocol |
H9 cells were synchronized in their cell cycle by incubating them with nocodazole (100 ng/ml) for 16h. Then, nocodazole was withdrawn and cells induced to differentiated into mesendoderm for 24h (In CDM-PVA medium supplemented with Insulin (7ug/ml), Activin A (100 ng/ml), BMP (10 ng/ml), LY294002 (10 mM ), and CHIR99021 (3 mM). To block cells in their S phase during differentiation, we added Hydroxyurea (200 mM) at the start of the differentiation. To block the cells in their G2/M phases during differentiation, we added nocodazole 8h after the induction of the differentiation. In this way, we made sure that the cells were able to re-initiate the cell cycle and get blocked in the following G2/M phase. After 24h of differentiation, cells were collected for RNA extraction.
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| Growth protocol |
H9 cells were grown in E8 medium containing penicillin (50 units/ml) and streptomycin (100 mg/ml)mycin, on plates coated with Vitronectin-XF (Stem Cells Technologies). Cells were incubated at 37ºC in atmosphere of 5% CO2
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNA extraction kit from Sigma, following manufaturer instructions.
RNA libraries were prepared for sequencing using Illumina TruSeq stranded mrNAseq kit; Ref N: 20020595
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Mesendoderm 24h + Nocodazole
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| Data processing |
Base calling was done using illumina package RTA v2.7.7, and instrument control package called HCS v3.4.0.38.
Adaptors were clipped before alignment and then re-inserted
Alignment files were converted into BAM files. Reads were counted using Rsubread package, based on protocol: "Analyzing RNA-seq data with DESeq2". Michael I. Love, Simon Anders, and Wolfgang Huber. (http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html)
Read counts were analyzed using DESeq2 based on protocole: "gene-level exploratory analysis and differential expression". Michael I. Love, Simon Anders, Vladislav Kim and Wolfgang Huber. (https://www.bioconductor.org/help/workflows/rnaseqGene/)
Genome_build: GRCh38_genecode
Supplementary_files_format_and_content: Read_counts_all_conditions.txt: Tab-delimited text file includes read counts per gene. DE_DESeq2_all_conditions.txt: Tab-delimited text file includes log2 fold change (log2FoldChange) and the adjustaed p-values (padj) for each convination of samples.
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| Submission date |
May 07, 2019 |
| Last update date |
May 31, 2021 |
| Contact name |
Rodrigo Andres Grandy |
| Organization name |
University of Cambridge
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| Department |
Surgery
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| Lab |
Vallier Lab
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| Street address |
Cambridge Stem Cell Institute/ Anne McLaren Laboratory West Forvie Building, Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0SZ |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE130829 |
Effect of Cell Division on Cell Differentiation (RNA-Seq H9 cells UD E24 E24HU E24Noc) |
| GSE130830 |
Effect of Cell Division on Cell Differentiation |
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| Relations |
| BioSample |
SAMN11587052 |
| SRA |
SRX5802462 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3754984_rna_seq_E24NocR1_sorted.bw |
235.9 Mb |
(ftp)(http) |
BW |
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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