|
| Status |
Public on Sep 14, 2023 |
| Title |
PER-403_taz_H3K27ac_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
PER-403
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PER-403
cell type: NUT carcinoma
treatment: 1 µM Tazemetostat
antibody: Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb (Cell Signaling Technology #8173, RRID: AB_10949503)
spike-in: Drosophila melanogaster Kc167 cells
|
| Treatment protocol |
Tazemetostat and ABBV-075 were solubilized in DMSO. Cells were treated with 1 µM tazemetostat (0.1% final DMSO) or 1 µM tazemetostat + 0.702 nM ABBV-075 (0.1% final DMSO) for 96 hours. After the first 48 hours of treatment, cells were retreated with fresh growth media and compounds.
|
| Growth protocol |
10-15 cells were grown in DMEM with 4.5 g/L glucose and sodium pyruvate without L-glutamine (Corning #15013CV) + 10% Fetal Bovine Serum (Atlanta Biologicals #S10350) + 1% GlutaMAX (Gibco #35050061) + 1% Penicillin-Streptomycin (Gibco #15140122) + 1.25 µg/mL Plasmocin prophylactic (Invivogen #ant-mpp) at 37° C with 5% CO2. PER-403 cells were grown in DMEM with 4.5 g/L glucose and sodium pyruvate without L-glutamine (Corning #15013CV) + 20% Fetal Bovine Serum (Atlanta Biologicals #S10350) + 1% GlutaMAX (Gibco #35050061) + 1% Penicillin-Streptomycin (Gibco #15140122) + 1.25 µg/mL Plasmocin prophylactic (Invivogen #ant-mpp) at 37° C with 5% CO2. Kc167 cells were grown in CCM3 Media (HyClone #SH30062.02) at 25° C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Human cells were mixed with Drosophila cells at a 4:1 ratio and then cells were bound to magnetic beads and permeabilized with digitonin. Permeabilized cells were incubated with the indicated antibody plus an anti-Drosophila Histone H2Av antibody (Active Motif Spike-in Antibody, #61686) overnight. Antibody-bound chromatin was released by in situ protein A-MNase digestion and the soluble DNA then purified.
DNA was prepared for next-generation sequencing using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Data processing |
Basecalling was performed using Illumina DRAGEN FASTQ Generation v3.7.4.
Reads were trimmed for adaptor sequences using trimmomatic v0.36.
Reads were aligned to concatenated hg38/Genome Reference Consortium Human Reference 38 (GRCh38) and dm6/BDGP Release 6 + ISO1 MT assemblies of the human and Drosophila melanogaster genomes, respectively, using using bowtie2 v2.3 with options --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700.
Properly paired, mapped reads with a MAPQ ≥ 30 were retained using samtools v1.3.
PCR duplicates were removed with picard MarkDuplicates v2.9.
deepTools v3.4 was used to compute signal tracks by running bamCoverage with parameters --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64 --scaleFactor $OR where the occupancy ratio ($OR) is used for spike-in normalization with D. melanogaster as the calibration genome, as previously described (Hu et al., 2015).
Epic2 v0.0 was used to identify broadly enriched domains with parameters -w 200 -g 5. IgG libraries were used as controls for domain annotation.
Called domains were filtered by signal strength and by domain size. Signal strength was determined by subtracting the rpm normalized input reads from rpm normalized sample reads for each called domain and subsequently ranked in increasing order of counts. The ROSE algorithm (Rank Ordering of Super-Enhancers) (Whyte et al., 2013) was applied to the ranked dataset to determine the cutoff threshold for signal strength by sliding a diagonal line and finding the point along the abscissa where the diagonal line was tangent to the rank-ordered curve. The cutoff for feature size was determined in a similar manner by ranking the domain length in base pairs and applying ROSE. Domains that exceeded both the signal cutoff and the size threshold were retained using bedtools intersect v2.30 with default parameters.
Often, we observed closely spaced domains and to span these domains and capture the entire domain, domains within 25 kb of one another were combined using bedtools v2.30 with command bedtools merge.
The above was performed for each biological replicate, and, then, to create the highest confidence domain list, domains identified in both replicates were merged using bedtools intersect v2.30 with default parameters. The resulting domains were considered “megadomains”. Epic2-identified domains that overlapped in each biological replicate but did not overlap with megadomains were considered "not Megadomains".
Assembly: GRCh38 concatenated with dm6/BDGP Release 6 + ISO1 MT
Supplementary files format and content: *.bw file of CUT&RUN track in bigWig format.
Supplementary files format and content: *.bed file of domain locations in bed format.
Library strategy: CUT&RUN
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| |
|
| Submission date |
Mar 30, 2023 |
| Last update date |
Sep 14, 2023 |
| Contact name |
Kyle Eagen |
| E-mail(s) |
[email protected]
|
| Organization name |
Baylor College of Medicine
|
| Department |
Department of Molecular and Cellular Biology
|
| Lab |
Eagen Lab
|
| Street address |
1 Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL30173 |
| Series (1) |
| GSE228533 |
CUT&RUN profiling of histone H3K27ac and H3K27me3 localization after pharmacological inhibition of EZH2 and BET proteins |
|
| Relations |
| BioSample |
SAMN33981305 |
| SRA |
SRX19815944 |
