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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jul 06, 2022 |
Title |
WT_mESC_1 |
Sample type |
SRA |
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Source name |
90X mouse embryonic stem cells and 10X yeast cells
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Organism |
Mus musculus |
Characteristics |
genotype: WT mESC cell type: embryonic stem cells molecule subtype: 4sU RNA (newly synthesized RNA)
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Growth protocol |
Mouse ES E14 cells were cultured on plates coated with 0.1% gelatine solution in 1x PBS using DMEM medium supplemented with 15% foetal calf serum ES-tested, 2 mM ʟ-glutamine, 0.1% β-mercaptoethanol, 100 UI/ml penicillin and 100 μg/ml streptomycin, 0.1 mM non-essential amino acids, 1500 U/ml leukaemia inhibitory factor, 3 μM CHIR99021 and 1 μM PD0325901. Cells were grown at 37°C with 5% CO2 levels. S. pombe cells were grown in autoclaved YES medium (yeast extract, adenine, histidine, uracil, leucine, lysine, 3% glucose) at 32°C.
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Extracted molecule |
total RNA |
Extraction protocol |
We purify newly transcribed RNA by adding 4sU (4-thiouridine), a metabolic analogue of uridine, to the cell culture medium for 15 minutes before collecting the cells. The cells readily take up 4sU and incorporate it into RNA transcribed during the time period of 15 minutes. This protocol is used to label newly transcribed RNA in both, human U2OS cells and mouse embryonic stem cells. 4tU was used to label newly synthesized RNA from S. pombe cells which served as a external spike-in for normalization. The incorporation of 4sU (with its specific thiol group) into newly transcribed RNA allows to separate the 4sU-containing RNA from the total RNA using biotinylation and Streptavidin-coated beads. The total RNA, used for the purification of the 4sU-containing RNA, was fragmented prior to purification (into on average ~700 bp fragments). For mESC, 4sU RNA-seq libraries were generated from 15 ng of purified, newly synthesized RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA, Cat# RS-122-2301) according to the Illumina protocol with the following modifications. 4sU-labelled RNA was cleaned up using 1.8X RNAClean XP beads and fragmented using divalent cations at 94°C for 1 minutes without depletion of rRNA. While, double stranded cDNA synthesis and adapter ligation were performed according to manufacturer instructions, the number of PCR cycles for library amplification was reduced to 10 cycles. After purification using SPRIselect beads (Beckman-Coulter, Villepinte, France, Cat# B23319), the libraries were sequenced with 1x 50 base pairs on a HiSeq4000 machine (Illumina).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
Very similar material as project S19062: The material is nascent, 4sU-labelled RNA purified using a biotinylation procedure from a mixture of 90X mouse embryonic stem cells and 10X yeast cells. The yeast RNA serves as a spike-in control. Prior to the purification of the nascent RNA, the RNA was fragmented using Covaris to an average RNA size of ~1 kb. No Ribozero required. VQFR25
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Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Reads were preprocessed using CUTADAPT 1.10 in order to remove adaptors and low-quality sequences and reads shorter than 40 bp were removed for further analysis. rRNA sequences were removed for further analysis. For samples VQFR25, VQFR26, VQFR29, VQFR30, remaining reads were aligned to a hybrid genome composed of mm10 and ASM294v2 assemblies of M. musculus and S. pombe genomes respectively with STAR 2.5.3a. For samples VQFR188, VQFR189, VQFR191, VQFR192, the hybrid genome was composed of hg38 and ASM294v2 assemblies of H. sapiens and S. pombe genomes respectively. Gene quantification was performed with htseq-count 0.6.1p1, using “union” mode and Ensembl 93 annotations for all organisms except for S. pombe where Ensembl Fungi 41 annotations were used. For 4sU-seq data, “type” option was set to “gene” in order to take also into account reads aligned onto introns. Differential gene expression analysis was performed using DESeq2 1.16.1 Bioconductor R package on H. sapiens or M. musculus counts normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber based on the spike-in counts (using the following options: cooksCutoff=TRUE, independentFiltering=TRUE, alpha=0.05). P-values were adjusted for multiple testing using the Benjamini and Hochberg method. Genome_build: hg38 for human data, mm10 for mouse data Supplementary_files_format_and_content: alldata*txt: raw counts, norm. counts, DESeq2 results Supplementary_files_format_and_content: spike*txt: raw counts of spike-in RNA from S. pombe
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Submission date |
Jun 01, 2021 |
Last update date |
Jul 06, 2022 |
Contact name |
Veronique Fischer |
Organization name |
IGBMC
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Street address |
1 Rue Laurent Fries
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City |
Illkirch CEDEX |
ZIP/Postal code |
67404 |
Country |
France |
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Platform ID |
GPL21103 |
Series (1) |
GSE175901 |
SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells |
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Relations |
BioSample |
SAMN19487771 |
SRA |
SRX11039357 |
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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