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| Status |
Public on Apr 25, 2019 |
| Title |
RING1Bfl_UNT_NucRNA_rep3 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila spike-in, untreated control, NucRNA-seq
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell line: Ring1a-/-;Ring1bfl/fl
replicate: 3
treatment agent: none
treatment time point: 0 hr
clone: B9
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
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| Treatment protocol |
To induce conditional removal of distinct PRC1 complexes or their combinations in Pcgf4-/-; Pcgf2fl/fl, Pcgf1fl/fl, Pcgf3/5fl/fl, Pcgf1/3/5fl/fl, Pcgf1/3/5/2fl/fl, Pcgf1/3/5/6fl/fl and Ring1a-/-; Ring1bfl/fl ESC lines, cells were treated with 800 nM 4-hydroxytamoxifen (TAM) for 72 hours.
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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).
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| Extracted molecule |
total RNA |
| Extraction protocol |
1 x 10^7 mouse ESCs were mixed with 4 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). To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was used to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 13-15 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to an average size of less than 1 kb.
RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) following manufacturer’s guidelines. Libraries from sonicated genomic DNA were constructed using NEBNext Ultra DNA Library Prep Kit for Illumina and indexing was done using NEBNext Multiplex Oligos, following manufacturer’s guidelines. Both RNA-seq and gDNA-seq libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicate.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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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). For gDNA-seq, paired-end reads were aligned to the genome sequence of concatenated mm10+dm6 genomes using Bowtie 2 with the “--no-mixed” and “--no-discordant” options specified. 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. To correct for any variation in spike-in cell mixing in different biological replicates, the ratio of dm6/mm10 total read counts was calculated from corresponding genomic DNA-seq samples and later used to adjust downsampling factors.
Biological triplicate read counts 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 derived from a corresponding Input or genomic DNA-seq sample. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5.
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files generated with genomeCoverageBed from BEDTools representing genome coverage for merged biological replicates which have been pre-normalised using spike-in downsampling factors
Supplementary_files_format_and_content: DESeq2 results table with normalised read counts, estimated log2 fold changes and statistical parameters of differential expression gene analysis
Supplementary_files_format_and_content: Tables with RefSeq IDs and MGI symbols for selected groups of genes described in the paper (related to Figures S5 and S6)
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| Submission date |
Sep 06, 2018 |
| Last update date |
Feb 21, 2020 |
| Contact name |
Nadezda A Fursova |
| E-mail(s) |
nfursova.msu@gmail.com
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| Organization name |
University of Oxford
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| Department |
Department of Biochemistry
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| Lab |
Klose lab
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| Street address |
South Parks Rd
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| City |
Oxford |
| ZIP/Postal code |
OX13QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE119619 |
Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression (RNA-Seq) |
| GSE119620 |
Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression |
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| Relations |
| BioSample |
SAMN09987802 |
| SRA |
SRX4651087 |
| 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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