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| Status |
Public on Jun 13, 2024 |
| Title |
TT_1h_washout_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: GSI 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, 11 15-cm dishes were seeded with 10 million SC2 cells per plate in SC2 media with 1 µM GSI. For each time course replicate (n=2), 10 samples were prepared: 0 hr mock washout + BRM014 (MedChem Express), 0 hr mock washout – BRM014 (reference condition in this text), 1 hr washout + BRM014, 1 hr washout - BRM014, 1 hr mock washout + BRM014, 1 hr mock washout - BRM014, 4 hr washout + BRM014, 4 hr washout - BRM014, 4 hr mock washout + BRM014, 4 hr mock washout - BRM014. The 11th plate was used as a sentinel plate: cells were harvested from the plate using 0.25% trypsin and were counted to determine the total number of cells per plate. For each replicate, it was assumed that all 10 experimental plates had the same number of cells as the sentinel plate. 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 |
nuclear RNA |
| Extraction protocol |
Washout and mock washouts were performed as described above in the GSI washout section. 1 µM BRM014 or the equivalent volume of DMSO was added in the replenishing media after the two media changes. Labeling was performed for 10 minutes in 15 mL of media supplemented with 500 µM 4-Thiouridine (4-SU) (Thermo Fisher J60679) with GSI and/or BRM014 depending on the experimental condition. For the 0 h timepoints, labeling media was added immediately after the mock washouts. For the 1 hr and 4 hr timepoints, labeling media was added at 50 min or 3 h and 50 min after washout, respectively. To harvest cells for TT-seq, plates were quickly rinsed with 20 mL PBS; then 2 mL of Trizol (Thermo Scientific15596026) was added to the plate. After 3 min of lysis in the dish, the cell lysate in Trizol was collected and frozen at -80°C. 1.4 mL of each sample was used to prepare RNA. For normalization purposes, fly spike-in cells were used. Fly (S2) cells used for spike-in were labeled for 2 hr with 4-SU (500 µM), and harvested in Trizol. 5 % 4-SU labeled spike-in cells (e.g. 0.5 million fly cells into 10 million human cells) were added directly to the Trizol lysate for each experimental condition. This was then split into two 700 µL tubes for purification. First the lysate was homogenized using a QiaShredder column (Qiagen 79656). 150 µL of chloroform was added to the homogenate, which was then vortexed, incubated for 5 minutes, and spun down at 12,000 g for 15 minutes at 4ºC to separate the phases. 360 μL of the aqueous phase was transferred to a new tube, and 540 µL of ethanol with 1.5 mM DTT was added. This material was then applied to miRNeasy mini kit column (Qiagen 217004), and RNA was isolated following the kit protocol, using an on-column digestion with DNase (Qiagen 79254). After the isolation, the two columns per sample were re-combined. Aliquots of RNA were removed for quantification by spectrophotometry and analysis of RNA integrity by Agilent TapeStation 4200 using RNA high sensitivity tapes. All samples were confirmed to have RNA integrity numbers (RIN) > 9.0 before proceeding to TT-seq library construction.
50 µg of RNA per sample was brought to a volume of 80 µL with nuclease-free water and placed on ice. RNA was then lightly fragmented by addition of 20 µL cold 5X fragmentation solution (375 mM Tris-HCl (pH 8.3), 562.5 mM KCl, 22.5 mM MgCl2) and incubation at 94°C for 2 minutes 15 seconds. At the end of the fragmentation time, RNA was placed immediately on ice and 25 µL of cold 250 mM EDTA was added. RNA was precipitated by addition of 1/10 volume of 5 M NaCl, 2.5 volumes of 100 % ethanol and incubation at -20°C overnight. RNA was pelleted, washed, quantified, and analyzed again as above. Fragmented RNA was biotinylated essentially as described in Duffy et al.36 with the following modifications: the biotinylation reaction was performed in a total volume of 200 µL and allowed to incubate for 45 min in the dark. Excess biotin was removed using chloroform:isoamyl alcohol as per Dölken et al.37 and MaXtract tubes (Qiagen 129056) were used to separate organic and aqueous phases. Biotinylated RNA was resuspended in 100 µL of nuclease-free water and aliquots taken to use as the total RNA input fraction. In parallel, Dynabeads M-280 Streptavidin (ThermoFisher 11205D) were prepared for binding to render them RNase-free: for each sample, 75 µL of beads were used and treated in batch to render them RNase free. The beads were incubated 10min in a solution of 100 mM NaOH, 50 mM NaCl, placed on a magnetic stand, and then washed, resuspending the beads fully for each wash, twice with 500 µL of 100 mM NaCl, twice with 1 X TT-seq wash solution (100 mM Tris-HCl (pH 7.5), 10mM EDTA, 1 M NaCl, 0.05% Tween 20 in nuclease free water to which 1 µL SUPERase-In RNase Inhibitor (ThermoFisher AM2696) per 5mL solution is added prior to use), once in 0.3 X TT-seq wash solution, and finally resuspended in 52 µL/sample of 0.3 X TT-seq wash solution + 1 µL/sample SuperaseIn RNase Inhibitor.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
sorted_1h_washout_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_1h_washout_BRM014_merged_depthnormalized_minus_single.bedGraph.bw
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| Data processing |
*library strategy: Transient transcriptome sequencing (TT-seq)
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. Reads were first mapped to the spike genome (dm6) using bowtie1.2.2 (-n2 -l 40 -X1000 -p 5 --best -3 1 parameters). Reads not mapping to the spike genome were mapped to the human genome (hg38) using with STAR2.7.3a40, using parameters --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 42949672960 --outMultimapperOrder Random --outSAMattrIHstart 0 --outFilterType BySJout --outFilterMismatchNmax 4 --alignSJoverhangMin 8 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outWigType bedGraph --outWigNorm None --outFilterScoreMinOverLread 0 –outFilterMatchNminOverLread 0. Duplicates were also removed using STAR, and stranded coverage bedGraph files were generated from deduplicated BAM files using STAR. To normalize samples, reads mapping to exons in the active gene models were counted using featurecounts, and used to calculate size factors using DESeq2. 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
Supplementary files format and content: excel file with read counts per gene for all samples
Library strategy: TT-seq
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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) |
| GSE269116 |
Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility [TT-seq] |
| GSE269128 |
Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility. |
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| Relations |
| BioSample |
SAMN41686649 |
| SRA |
SRX24807811 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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