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Status |
Public on Jul 06, 2022 |
Title |
U2OS_1 |
Sample type |
SRA |
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Source name |
U2OS cells
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Organism |
Homo sapiens |
Characteristics |
genotype: U2OS parental cell line: U2OS molecule subtype: 4sU RNA (newly synthesized RNA)
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Growth protocol |
Human U2OS osteosarcoma cells (HTB-96; ATCC) were cultured using DMEM medium supplemented with 10% foetal calf serum (Sigma Aldrich, Cat# F7524) and 40 µg/ml gentamycin (KALYS, Cat# G0124-25). Cells were grown at 37°C with 5% CO2 levels.
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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 U2OS cells, 4sU RNA-Seq libraries were generated from 15ng of purified, newly synthesized RNA using Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit and IDT for Illumina RNA UD Indexes, Ligation (Illumina, San Diego, USA, Cat# 20040525 and 20040553/4, respectively), according to manufacturer's instructions. Briefly, abundant ribosomal RNAs were depleted by hybridization to specific DNA probes and enzymatic digestion. The depleted RNAs were purified and fragmented using divalent cations at 94°C for 2 minutes. After random hexamers annealing, fragmented RNAs were then reverse transcribed into first strand complementary DNA (cDNA). Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France, Cat# B23319) and the final libraries were checked for quality and quantified using capillary electrophoresis.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
VQFR188
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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 |
GPL20301 |
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 |
SAMN19487775 |
SRA |
SRX11039353 |
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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