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| Status |
Public on Jun 13, 2024 |
| Title |
ATAC_1h_GSI_BRM014_rep2 |
| Sample type |
SRA |
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| Source name |
SC2
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SC2
cell type: squamous cell carcinoma
treatment: mock washout
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| Treatment protocol |
For all gamma secretase inhibitor (GSI) washouts, media was removed, cells were washed twice with media containing 0.1% DMSO instead of GSI, and replenished with media containing 0.1% DMSO instead of GSI, for a total of three media changes. In the mock washouts, both the washing media and the replenishing media contained 1 μM GSI. For experiments with BRM014 (MedChem Express HY-119374), the replenishing media, but not the washout media, either contained 1µM BRM014 or 0.01% DMSO depending on the sample. The evening before the time course, cells were plated in 12 well dishes, at 125,000 cells / well in SC2 media with 1 µM GSI. For each time course, 14 samples were prepared: 0 hr mock washout + BRM014, 0 hr mock washout – BRM014, 1 hr washout + BRM014, 1 hr washout - BRM014, 1 hr mock washout + BRM014, 1 hr mock washout - BRM014, 2 hr washout + BRM014, 2 hr washout - BRM014, 2 hr mock washout + BRM014, 2 hr mock washout - BRM014, 4 hr washout + BRM014, 4 hr washout - BRM014, 4 hr mock washout + BRM014, 4 hr mock washout - BRM014. Replicates were performed on different days (n=2).
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| Growth protocol |
SC2 squamous cell carcinoma cells were cultured in keratinocyte media, consisting of: 3:1 DMEM:F12 + 10% Fetal Bovine Serum (Gemini Bio-Products 100-106) + 1X Penicillin-Streptomycin (ThermoFisher 15140163) + 1X Reagent Mix (see below) + 1 μM Compound E (EMD Millipore565790) gamma secretase inhibitor (GSI). 100X Reagent Mix was prepared in 3:1 DMEM:F12 media, containing 40 mg/mL Hydrocortisone (Sigma-Aldrich H0888), 500 ng/mL Insulin (Sigma-Aldrich I6634), 1mg/mL EGF (Sigma-Aldrich E9644), 0.84 mg/mL Cholera toxin (Sigma-Aldrich C9903), 500 mg/mL Transferrin (Sigma-Aldrich T2036), and 1.3 mg/mL Liothyronine (Sigma-Aldrich T6397). GSI was prepared as a 1 mM stock of Compound E in DMSO (Sigma-Aldrich 472301). Cells were split every 3-4 days, using 0.25% trypsin (VWR 45000-664) supplemented with 1 μM GSI. Cells were incubated in 0.25% trypsin at 37 ºC until cells began to slough off the plate, then were quenched with fresh media and replated. Cells were routinely tested for mycoplasma, and used for experiments within 1 month of thawing a fresh vial.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At the time point after washout or mock washout, cells were rinsed in PBS, and harvested using 0.25 % trypsin with GSI and/or BRM014 according to the sample, and quenched with SC2 media containing GSI and/or BRM014. Cells were counted, and 100,000 cells were transferred to an Eppendorf tube, and combined with 10,000 fly spike-in cells. The spike-in S2 cells had been collected, resuspended in BAMBANKER cryopreservative media (VWR 101974-112), aliquoted, and stored at -80°C. All spike in aliquots for one experimental time course replicate were thawed and combined before beginning the time course. ATAC was performed according to the OmniATAC protocol. Briefly, cells were pelleted at 500 g for 5 min at 4°C, and supernatant was removed. The cell pellet was resuspended in 50 mL ATAC lysis buffer by pipetting up and down 3 times (Cold ATAC-Resuspension Buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2) + 0.1 % NP40 (EMD Millipore 492018) + 0.1 % Tween-20 (EMD Millipore 655206) + 0.01 % digitonin (Promega G9441)). Up to 4 samples were processed at 1 time, and samples were staggered by 15 seconds. After exactly 3 min of lysis, 1 mL of ATAC wash buffer (Cold ATAC-Resuspension Buffer + 0.1 % Tween-20 (EMD Millipore655206)) was added to the tube. Nuclei were pelleted at 500 g for 10 min at 4°C, and supernatant was removed. Nuclei were resuspended in 50 mL of Transposition mix (1x TD buffer (Illumina 20334197), 3 mL Tn5 transposase (Illumina 20334197), 0.33x PBS, 0.01 % digitonin, 0.1 % Tween-20), and incubated at 37°C in a thermomixer at 1000 rpm for 30 minutes. The reactions were quenched with 250 mL of binding buffer from the MinElute PCR purification kit (Qiagen 28004) and purified using this kit.
The eluate was then used as the input for barcoding PCR, to add sequencing adapters and sequencing barcodes, as described in Grandi et al.. Both the qPCR and Qubit methods were used to quantify the libraries to determine the total number of PCR cycles, as described in the OmniATAC protocol. After the final PCR cycles, libraries were cleaned up with the MinElute PCR purification kit, quantified using the NEBNext Library Quant kit (New England Biolabs E7630), and pooled for sequencing. Libraries were sequenced paired end on a NovaSeq with an S1 single lane 100 cycle kit.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
merged_ATAC_single_1_gsi_brm014.bedGraph.bw
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| Data processing |
Read pairs were trimmed to 100 base pairs using a custom script (trim_and_filter_PE.pl, https://doi.org/10.5281/zenodo.11222086), and reads with a minimum average quality score of 20 were kept. Adapter sequences were removed using cutadapt 1.1441. Reads were first mapped to the spike genome (dm6), using bowtie1.2.2 (-k1 -v2 -X1000 – allow-contain -- best parameters). Reads that did not map to the spike genome were mapped to the human genome (hg38), using bowtie1.2.245 (-k1 -v2 -X1000 – allow-contain -- best parameters). Samtools 1.943 was used to flag duplicate reads, which were then removed. A custom script, extract_fragments.pl, was used to filter and retain unique reads between 10 and 150 base pairs, which correspond to regions of open chromatin, and convert files into bedGraph format. Spike return rates were not significantly different between samples. To normalize samples, reads mapping to the -1 kb to +1 kb region around active promoters in this cell type were counted using the custom script makeheatmap with the “-b v -v t -s b” options. The number of these reads was used to calculate size factors using DESeq246. These size factors were scaled so the minimum was 1, and used to normalize bedGraphs with the custom script normalize_bedGraph.pl. Biological replicates (n=2) were merged using the script bedgraphs2stbedGraph.pl.
Assembly: hg38
Supplementary files format and content: bigwig files with replicates merged for each time point
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| Submission date |
Jun 05, 2024 |
| Last update date |
Jun 13, 2024 |
| Contact name |
Julia M Rogers |
| E-mail(s) |
julia_rogers@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
BCMP
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| Lab |
Blacklow Lab
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| Street address |
45 Shattuck Street
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE269112 |
Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility [ATAC-seq] |
| GSE269128 |
Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility. |
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| Relations |
| BioSample |
SAMN41686523 |
| SRA |
SRX24807546 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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