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| Status |
Public on May 03, 2021 |
| Title |
Bap1flfl_ATAC.Input_OHT_rep4 |
| 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: Bap1fl/fl
cell type: mouse embryonic stem cells (mESCs)
genotype: Bap1fl/fl; Rosa26::ERT2-Cre
clone: 7-5;7-2
strain: E14TG2a
replicate: 4
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, Bap1fl/fl mESCs were treated with 800 nM 4-hydroxytamoxifen (OHT) for 96 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 cATAC-seq, 1×107 ESCs (untreated and OHT-treated) were mixed with 4×106 Drosophila SG4 cells in 1x PBS and then lysed 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. Nuclei were recovered by centrifugation at 1000 g for 5 min at 4°C and washed three times in ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2). The concentration and integrity of nuclei were assessed using 0.4% Trypan Blue staining (ThermoScientific). Next, 5×105 nuclei were resuspended in Tn5 reaction buffer (10 mM TAPS, 5 mM MgCl2, 10% dimethylformamide) and incubated with Tn5 transposase (25 µM, generated in house as previously described (King and Klose 2017)) for 30 min at 37°C. Tagmented DNA was purified using MinElute columns (QIAGEN) and eluted in 10 μl elution buffer. To control for the Tn5 transposase sequence bias and to determine the exact spike-in ratio for each individual sample, 50 ng of genomic DNA, isolated from the same nuclei preparation by phenol-chloroform extraction, was tagmented with Tn5 transposase (25 µM) for 30 min at 55°C and purified using MinElute columns (QIAGEN).
Libraries for cATAC-seq and gDNA-seq were prepared by PCR amplification using the NEBNext High-Fidelity 2X PCR Master Mix and custom-made Illumina barcodes (Buenrostro et al. 2015). Libraries were purified by two rounds of Agencourt AMPure XP bead cleanup (1.5× bead:sample ratio). 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). Libraries were sequenced using the Illumina NextSeq 500 platform in biological quadruplicate using 80 bp paired-end reads.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
BAP1ff_Input_TAM_rep5_S16
processed data file:
mESC_BAP1ff_TAM_ATAC.Input_mm10.UniqMapped_sorted_rmdup_filtered_downsampled_MERGED.Coverage.bw
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| Data processing |
For cATAC-seq and gDNA-seq, Bowtie 2 was used to align paired-end reads to the concatenated mouse and spike-in genome sequences (mm10+dm6) with the “--no-mixed” and “--no-discordant” options. Non-uniquely mapping reads were discarded, and PCR duplicates were removed with Sambamba. Reads mapping to the mitochondrial chromosome and other genomic regions with artificially high counts or low mappability, derived from the ENCODE blacklist, were also discarded.
For visualization of cATAC-seq, the data were internally calibrated using dm6 spike-in as described previously (Turberfield et al. 2019). Briefly, uniquely aligned mm10 reads after filtering 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 TSS ± 2.5 kb genomic regions were compared for individual biological replicates using multiBamSummary and plotCorrelation from deepTools. For each experimental condition, biological replicates correlated well (Pearson correlation coefficient > 0.9) and were merged for downstream analysis.
Genome_build: mm10; dm6
Supplementary_files_format_and_content: BigWig files were generated using the bamCoverage function from deeptools representing genome coverage of merged spike-in normalised biological replicates.
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| Submission date |
Nov 23, 2020 |
| Last update date |
May 03, 2021 |
| Contact name |
Nadezda A Fursova |
| E-mail(s) |
[email protected]
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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) |
| GSE161994 |
BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome [ATAC-seq] |
| GSE161996 |
BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome. |
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| Relations |
| BioSample |
SAMN16873256 |
| SRA |
SRX9555340 |
| 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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