GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4949993 Query DataSets for GSM4949993
Status Public on Sep 21, 2023
Title MCF10A p53 R248Q mutant cells - RNA-seq
Sample type SRA
Source name ectopic expression of p53 R248Q mutant protein with V5 tag at C-term
Organism Homo sapiens
Characteristics cell type: MCF10A mammary epithelial cells
genotype/variation: p53 R248Q mutant cells
treatment: untreated
cell line: MCF10A
Treatment protocol No treatment
Growth protocol MCF10A cells were cultured at 37˚C in a 5% CO2 humidified incubator. DMEM/F12 media (ThermoFisher #11320-082) was supplemented with 5% Horse Serum (ThermoFisher #16050-122), hEGF (10ng/ml, ThermoFisher #PHG0311), hydrocortisone (0.5µg/ml, Sigma #H0888), cholera toxin (100ng/ml, Sigma #C8052), and Insulin (10µg/ml, Sigma #I9278). Cell were routinely passaged with trypsin (0.25% in HBSS with 0.2g/ml EDTA, GE Healthcare #SH30042.01) at 80-90% confluence.
Extracted molecule total RNA
Extraction protocol RNA-Seq: Total RNA extracted from the cells (Qiagen cat no. 74134) were used to prepare cDNA using Nugen’s Ovation RNA-Seq System via single primer isothermal amplification (Catalogue # 7102-A01) automated on the Apollo 324 liquid handler from Wafergen.
RNA-Seq: cDNA was sheared to approximately 300 bp fragments using the Covaris M220 ultrasonicator. Libraries were generated using Kapa Biosystem’s library preparation kit (KK8201). Fragments were end-repaired and A-tailed, individual indexes and adapters (Bioo, catalogue #520999) were ligated on each separate sample. The adapter ligated molecules were cleaned using AMPure beads (Agencourt Bioscience/Beckman Coulter, A63883), and amplified with Kapa’s HIFI enzyme. The library was then analyzed on an Agilent Bioanalyzer, and quantified by qPCR (KAPA Library Quantification Kit, KK4835) before multiplex pooling and sequencing a 2x75 PE flow cell on the NextSeq500 platform (Illumina) at the ASU’s CLAS Genomics Core facility.
RNA-Seq: The library was then analyzed on an Agilent Bioanalyzer, and quantified by qPCR (KAPA Library Quantification Kit, KK4835) before multiplex pooling and sequencing a 2x75 PE flow cell on the NextSeq500 platform (Illumina) at the ASU’s CLAS Genomics Core facility.
chip-seq: All the MCF10A cell lines were fixed with 1% formaldehyde in PBS and incubated for 10 min at RT. Formaldehyde was quenched with glycine (125mM final conc.) for 5 min at RT. Cells were rinsed with ice cold PBS twice and scraped with 2ml of cold PBS with protease inhibitor (Sigma Aldrich Cat no. 04693124001). Cells were washed two more times with cold PBS with protease inhibitor and pellets from 20 million cells were snap frozen and stored at -80oC. 1ml of ChIP lysis buffer (1% SDS,10mM EDTA, 50mM Tris-HCl pH8.1) with protease inhibitors was added and the cells were sonicated in the Covaris M220 ultrasonicator for 8 min. The time and setting for sonication would vary with the cell-line and concentration. The lysates were spun at maximum speed at 4oC for 10 min and the DNA concentration was checked. The supernatant was transferred into fresh tube. 25-50ul of the lysate was incubated at 65oC overnight with 0.1M NaCl and run on a 2% agarose gel to check the sonication efficiency. You should get an average fragmentation of ~500 bp. For IP, 30ul of beads (Thermo Cat no. 11203D) were first washed with 1ml of cold BSA (5mg/ml) in PBS at RT (3 times). Then V5 antibody (CST Cat no. 13202S) was added to the beads in 1ml of PBS+ BSA. The beads with the antibody were incubated for 4 to 6 hrs at 4oC. The IP was performed on 50ug of total DNA and the samples were diluted accordingly in the dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0). The diluted chromatin was precleared with 15ul of washed beads for 1hr at 4oC. 5% of this precleared lysate was kept as input control. PBS/BSA was aspirated from the antibody coated beads and precleared lysate was added to incubate O/N at 4oC on a rotating wheel. Next day beads were collected using the magnetic concentrator and washed 6 times with ChIP RIPA buffer (50mM HEPES, 1mM EDTA, 0.7% Na Deoxycholate, 1% NP-40, 0.5M LiCl pH 7.6) at RT on a rotating platform for 10 min between every wash. Then washed twice with 1X TE (pH7.6) at RT. 100 ul of Elution buffer (1% SDS, 0.1M NaHCO3, 0.1M NaCl) was added to the beads and input samples (make up volume to 100ul). The beads were vortexed in this solution every few minutes for 30 minutes in total at RT and incubated at 65oC O/N for 12-14 hrs. Next day 1ul of proteinase K (10mg/ml) was added and incubated at 42oC for 2 hrs. The IP DNA was purified with QIAquick PCR purification kit.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing STAR (V020201) (Dobin 2013) was used to align reads to the human genome (GRCh38.p92/hg38) to counts with the following configuration: outSAMmultNmax 1 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outFilterMultimapNmax 1 --alignSJoverhangMin 6 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.2 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000
Mitochondrial reads were removed before gene counts log2 TPM (transcripts per million reads) normalization. Transcript length used in TPM normalization was calculated using the longest transcript of each gene
csv file of gene expression from individual cell lines. Score represent the gene expression level in log2(TPM+1)
Submission date Nov 30, 2020
Last update date Sep 22, 2023
Contact name Jin G Park
Organization name Arizona State University
Street address 727 E. Tyler St., Suite A220C
City Tempe
State/province AZ
ZIP/Postal code 85281-2115
Country USA
Platform ID GPL18573
Series (1)
GSE162341 Systematic phenotypic characterization and integrated analysis show heterogeneous neomorphic activities of different missense mutant p53 proteins
BioSample SAMN16956248
SRA SRX9605778

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap