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| Scope: Self Platform Samples Series Family Format: HTML SOFT MINiML Amount: Brief Quick GEO accession: |  |
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| Sample GSM4227187 | Query DataSets for GSM4227187 |
| | Status | Public on Dec 18, 2020 | | Title | MCF10A, WTp53 cDNA insert, DMSO, Replicate 1 | | Sample type | SRA | | Source name | MCF10A Cells | | Organism | Homo sapiens | | Characteristics | cell line: MCF10Atissue: mammary gland; breastcell type: epithelialcrispr/cas9 insertion: 2820bp: full length p53 cDNA followed IRES expressed mCherry followed by SV40 polyA sequnececrispr/cas9 insertion location: hg38 chr17:7,676,591treatment: %0.01 DMSO | | Treatment protocol | MCF10A WTp53 cells were seeded on three 25cm dishes (1x107 cells per dish) for each treatment 24 hours prior to the experiments ( 70% confluency at the time of the experiment). Cells were treated simultaneously with 10μM Nutlin3a or 0.1% DMSO for 1 hour. | | Growth protocol | MCF10A cells cultured in DMEM/F12 (Invitrogen #11330-032) media containing 5% horse serum (LifeTech #16050- 122), 20ng/mL EGF ((Peprotech #AF-100-15), 0.5μg/mL Hydrocortisone (Sigma #H0888-1g), 100ng/mL Cholera toxin (Sigma #C8052-2mg), 10μg/mL insulin (Sigma #I1882-200mg), and 1x Gibco 100x Antibiotic-Antimycotic (Fisher Sci, 15240062) penicillin-streptomycin. | | Extracted molecule | total RNA | | Extraction protocol | After treatment, cells were washed 3x with ice cold PBS, and then treated with 10 ml (per 15 cm plate) ice-cold lysis buffer (10 mM Tris–HCl pH 7.4, 2 mM MgCl2, 3 mM CaCl2, 0.5% NP-40, 10% glycerol, 1 mM DTT, 1x Protease Inhibitors (1mM Benzamidine (Sigma B6506-100G), 1mM Sodium Metabisulfite (Sigma 255556-100G), 0.25mM Phenylmethylsulfonyl Fluoride (American Bioanalytical AB01620), and 4U/mL SUPERase-In). Cells were centrifuged with a fixed-angle rotor at 1000xg for 15 min at 4◦C. Supernatant was removed and pellet was resuspended in 1.5 mL lysis buffer to a homogenous mixture by pipetting 20-30X before adding another 8.5 mL lysis buffer. Suspension was centrifuged with a fixed-angle rotor at 1000xg for 15 min at 4◦C. Supernatant was removed and pellet was resuspended in 1 mL of lysis buffer and transferred to a 1.7 mL pre-lubricated tube (Costar cat. No. 3207). Suspensions were then pelleted in a microcentrifuge at 1000xg for 5 min at 4◦C. Next, supernatant was removed and pellets were resuspended in 500 μL of freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 4U/ml SUPERase-In). Nuclei were centrifuged 2000xg for 2 min at 4◦C. Pellets were resuspended in 100 μL freezing buffer. To determine concentration, nuclei were counted from 1 μL of suspension and freezing buffer was added to generate 100 μL aliquots of 10x106 nuclei. Aliquots were flash frozen in liquid nitrogen and stored at −80◦C.Nuclear run-on experiments were performed as described previously (Mahat, D. B., Kwak, H., Booth, G. T., Jonkers, I. H., Danko, C. G., Patel, R. K., et al. (2016). Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nature Protocols, 11(8), 1455–1476. with the following modifications: the final concentration of non-biotinylated CTP was raised from 0.25 μM to 25 μM, a clean-up and size selection was performed using 1X AMPure XP beads (1:1 ratio) (Beckman) prior to test PCR and final PCR amplification, and the final library clean-up and size selection was accomplished using 1X AMPure XP beads (1:1 ratio) (Beckman). http://doi.org/10.1093/bioinformatics/btq033) with the following modification. The final concentration of non-biotinylated CTP was raised from 0.25uM to 25uM, and the final library clean up and size selection was accomplished using 1X AMPure XP (Beckman).PRO-seq | | Library strategy | OTHER | | Library source | transcriptomic | | Library selection | other | | Instrument model | Illumina NextSeq 500 | | Description | nascent RNAnascent RNA (PRO-seq) from MCF10A cells | | Data processing | processing of all sequencing data was performed using the NascentFlow Pipeline (doi: 10.17605/OSF.IO/NDHJ2)Genome_build: GRCh38Supplementary_files_format_and_content: bed | | Submission date | Dec 20, 2019 | | Last update date | Apr 20, 2022 | | Contact name | Dylan J Taatjes | | E-mail(s) | [email protected] | | Phone | 303 492-6929 | | Organization name | University of Colorado Boulder | | Department | Biochemistry | | Lab | Taatjes Lab | | Street address | Campus Box 596 | | City | Boulder | | State/province | CO | | ZIP/Postal code | 80303-0596 | | Country | USA | | Platform ID | GPL18573 | | Series (1) | | GSE142419 | Transcription factor enrichment analysis (TFEA): Quantifying the activity of hundreds of TFs from a single experiment |
| | Relations | | BioSample | SAMN13649813 |
| Supplementary file | Size | Download | File type/resource | | GSM4227187_7_DMSO_1hr_R1.tfit_bidirs.bed.gz | 306.4 Kb | (ftp)(http) | BED | | Processed data provided as supplementary file | | Raw data not provided for this record |
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