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| Status |
Public on May 03, 2021 |
| Title |
PRC1CPM_Bap1flfl_gDNAseq_OHT_rep3 |
| Sample type |
SRA |
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| Source name |
mESCs with Drosophila spike-in, tamoxifen-treated (96hr OHT), gDNA-seq
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell line: PRC1CPM;Bap1fl/fl
cell type: mouse embryonic stem cells (mESCs)
genotype: Ring1aI50A/D53K; Ring1b(WT->I53A/D56K)fl/fl; Bap1fl/fl; Rosa26::ERT2-Cre
clone: F6
strain: E14TG2a
replicate: 3
treatment agent: tamoxifen (OHT)
treatment time point: 96 hr
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
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| Treatment protocol |
To induce conditional removal of BAP1, on its own or in combination with PRC1 catalytic activity, Bap1fl/fl and PRC1CPM;Bap1fl/fl cells were treated with 800 nM 4-hydroxytamoxifen (OHT) for 96 hr. To induce conditional removal of PRC1 catalytic activity on its own, PRC1CPM cells were treated with 800 nM OHT for 72 hr.
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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 |
genomic DNA |
| Extraction protocol |
For cnRNA-seq, 1×10^7 ESCs (untreated and OHT-treated) were mixed with 4×10^6 Drosophila SG4 cells in 1x 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, 1xPIC (Roche)) for 1 min at room temperature, and 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, 3 mM MgCl2). Nuclei integrity was assessed using 0.4% Trypan Blue staining (ThermoScientific). Pelleted 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) to remove any contaminating DNA. Quality of RNA was assessed using the 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). RNA samples were depleted of rRNA with the NEBNext rRNA Depletion kit (NEB) prior to library prep. To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was saved to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 15 min using the BioRuptor Pico (Diagenode), shearing genomic DNA to an average size of less than 1 kb.
cnRNA-seq libraries were prepared using the NEBNext Ultra (for Bap1fl/fl and PRC1CPM) or Ultra II (for PRC1CPM;Bap1fl/fl) Directional RNA Library Prep kit (NEB). Libraries from sonicated genomic DNA were constructed using NEBNext Ultra (for Bap1fl/fl and PRC1CPM) or Ultra II (for PRC1CPM;Bap1fl/fl) DNA Library Prep Kit for Illumina, following manufacturer’s guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Both cnRNA-seq and gDNA-seq libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicates.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
PRC1CPM_BAP1ff_gDNAseq_Rep4_TAM_S18
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| Data processing |
For cnRNA-seq, first, paired-end reads were aligned using Bowtie 2 (with “--very-fast”, “--no-mixed” and “--no-discordant” options) against the concatenated mm10 and dm6 rDNA genomic sequence (GenBank: BK000964.3 and M21017.1) and reads mapping to rDNA were discarded. All unmapped reads were then aligned against the concatenated mm10 and dm6 genome sequences using STAR. Finally, reads that 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) to improve mapping of introns. Uniquely aligned reads from the last two steps were combined for further analysis. PCR duplicates were removed using Sambamba. For the corresponding gDNA-seq experiments, paired-end reads were aligned to the concatenated mm10+dm6 genome using Bowtie 2 with the “--no-mixed” and “--no-discordant” options. Only uniquely mapped reads after removal of PCR duplicates with Sambamba were used for downstream analysis.
For visualisation of cnRNA-seq, uniquely aligned mouse reads were internally calibrated using dm6 spike-in as described previously (Fursova et al. 2019). Briefly, mm10 reads were randomly subsampled based on the total number of spike-in (dm6) reads in each sample. To account for variations in the spike-in cell mixing, we used the ratio of spike-in/mouse total read counts in the corresponding gDNA-seq samples to correct the subsampling factors. After normalisation, read coverages across gene bodies were compared for individual biological replicates using multiBamSummary and plotCorrelation from deepTools. For each experimental condition, biological replicates corelated well (Pearson correlation coefficient > 0.9) and were merged for downstream analysis.
For differential gene expression analysis, read counts from individual biological replicates prior to spike-in normalisation were obtained. A custom Perl script utilising SAMtools was used to annotate a custom-built non-redundant mm10 gene set (n = 20633) with read counts. The non-redundant gene set was derived from mm10 refGene genes by removing very short genes with poor sequence mappability and highly similar transcripts.
To identify significant gene expression changes, we used a custom R script that incorporates spike-in calibration into DESeq2 analysis as described previously (Fursova et al. 2019). Briefly, dm6 read counts were obtained for unique dm6 refGene genes to calculate DESeq2 size factors for normalisation of raw mm10 read counts for a custom non-redundant mm10 gene set. Prior to quantification, dm6 reads were pre-normalised using the dm6/mm10 total read ratio in the corresponding gDNA-seq samples in order to account for variations in spike-in cell mixing. For visualisation and ranking of the effect sizes, we performed shrinking of log2-fold changes using the original DESeq2 shrinkage estimator with an adaptive normal distribution as prior. 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, and statistical significance levels for a non-redundant set of mm10 refGene genes.
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| Submission date |
Nov 23, 2020 |
| Last update date |
May 03, 2021 |
| 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) |
| GSE161995 |
BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome [RNA-seq] |
| GSE161996 |
BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome. |
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| Relations |
| BioSample |
SAMN16873209 |
| SRA |
SRX9555376 |
| 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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