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| Status |
Public on May 23, 2022 |
| Title |
Pcgf4-/-Pcgf2flfl_ESC_TAM_rep4 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila SG4 cells spike-in, tamoxifen-treated, Pcgf4-/-Pcgf2 fl/fl
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
tag: Pcgf4-/-Pcgf2 flfl
strain: Rosa26::ERT2-Cre
treatment: tamoxifen
treatment time: 72h
additional treatment: none
additional treatment time: none
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
protocol: calibrated nuclear RNA-seq
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| Treatment protocol |
To induce conditional removal of CDK-MED or cPRC1, cells were treated with 800 nM 4-hydroxytamoxifen (TAM, Sigma) for 96 hours (Med13/13l fl/fl) or 72h (Pcgf4-/-Pcgf2fl/fl). To induce differentiation of ES cells, cells were treated with 1 µM retinoic acid (RA, Sigma-Aldrich)
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| Growth protocol |
Mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor. ). Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin solution (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech). For RA differentiation, 4x10^6 ES cells were allowed to attach to gelatinised 15cm dishes for 6-8 hours and treated with 1 µM retinoic acid (Sigma-Aldrich) in EC-10 medium (DMEM supplemented with 10% fetal bovine serum, L-Glutamine, beta-mercaptoethanol, non-essential amino acids and penicillin/streptomycin) for 48 hours.
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| Extracted molecule |
total RNA |
| Extraction protocol |
2 x 10^7 mouse ESCs were mixed with 8 x 10^6 Drosophila SG4 cells in PBS. Nuclei were isolated in 1 ml HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05 % NP40 (IGEPAL CA630)), 1 mM PMSF, 3 mM DTT) for 1 min at room temperature. They were then recovered by centrifugation at 1000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris (pH 8.0), 3 mM MgCl2). Nuclei integrity was assessed using 0.4% Trypan Blue staining (ThermoScientific). Next, nuclei were resuspended in 1 ml of TRIzol reagent (ThermoScientific) and RNA was extracted according to the manufacturer’s protocol, followed by treatment with the TURBO DNA-free Kit (ThermoScientific). Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent) and high-quality RNA samples were depleted of rRNA using the NEBNext rRNA Depletion kit (NEB).
RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) following manufacturer’s guidelines. RNA-seq were sequenced as 80 bp (Med13/13lflfl) or 40 bp (Pcgf4-/-Pcgf2flfl) paired-end reads on the Illumina NextSeq 500 platform in biological quadruplicates
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Pcgf2fl_nucRNASeq_ESC_TAM_mm10_downsampledSpikeInInp_MERGED_FORWARD.bw
Pcgf2fl_nucRNASeq_ESC_TAM_mm10_downsampledSpikeInInp_MERGED_MinusStrand.bw
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| Data processing |
Paired-end reads were aligned using Bowtie 2 (with “--very-fast”, “--no-mixed” and “--no-discordant” options) against the concatenated mm10 and dm6 rRNA genomic sequence (GenBank: BK000964.3 and M21017.1), to filter out reads mapping to rRNA fragments. All unmapped reads from the first step, were aligned against the genome sequence of concatenated mm10 and dm6 genomes using the STAR RNA-seq aligner (Dobin et al. 2012). To improve mapping of intronic sequences for nascent transcripts abundant in nuclear RNA-seq, reads which failed to map using STAR were aligned against the mm10+dm6 concatenated genome using Bowtie 2 (with “--sensitive-local”, “--no-mixed” and “ --no-discordant” options). Reads that were mapped more than once were discarded. PCR duplicates were removed using SAMTools.
To internally calibrate RNA-seq, we spiked-in a fixed number of Drosophila SG4 to each experimental sample. For data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 reads in each sample.
Read counts from biological quadruplicates were determined using custom scripts utilising SAMTools for a custom-built, non-redundant mm10 gene set. Briefly, mm10 refGene genes were filtered to remove very short genes with poor sequence mappability and highly similar transcripts which resulted in the final set of 20,633 genes. Raw mm10 reads prior to spike-in normalisation were used for read counts quantitation for differential expression analysis.
The final set of 20,633 genes were used for differential analysis using a custom R script adapting DESeq2 (Love et al., 2014) for spike-in calibrated RNA-seq data. To incorporate spike-in calibration into this analysis, read counts for the spike-in genome at a control set of intervals were supplied to calculate DESeq2 size factors which were then used for DESeq2 normalisation of raw mm10 read counts. A set of unique dm6 refGene genes was used for spike-in normalisation of cnRNA-seq. Prior to quantitation, spike-in reads were pre-normalised to accurately reflect the actual spike-in ratio. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5.
Genome_build: mm10, dm6
Supplementary_files_format_and_content: Stranded bigWig files were generated using genomeCoverageBed from BEDTools representing genome coverage of merged spike-in normalised biological replicates.
Supplementary_files_format_and_content: DESeq2 results tables for differential gene expression analysis containing normalised read counts, raw and shrunk log2-fold changes (LFC), and statistical significance levels for a non-redundant set of mm10 refGene genes.
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| Submission date |
Oct 14, 2021 |
| Last update date |
May 23, 2022 |
| Contact name |
Emilia Dimitrova |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Rob Klose
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE185929 |
Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction (RNA-Seq) |
| GSE185930 |
Distinct roles of CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction |
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| Relations |
| BioSample |
SAMN22307032 |
| SRA |
SRX12622721 |
| 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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