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| Status |
Public on May 08, 2024 |
| Title |
Plasmid pool, replicate 2, DNA |
| Sample type |
SRA |
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| Source name |
na
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| Organism |
synthetic construct |
| Characteristics |
cell line: na
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| Treatment protocol |
10 µg of plasmid library were transfected per 5 mln cells via lipofection (Polyplus #101000046, #101000025). For MCF10A TAp63β inducible cell line and negative control (Gus) cell line, doxycycline was added at 500 ng/ml at the same time as transfection. Cells were harvested after 24h.
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| Growth protocol |
All human mammary epithelial cell lines MCF-10A TP53+/+ and TP53-/- (Sigma-Millipre clls1049) were cultured in 1:1 Dulbecco’s Modified Eagle’s Medium: Ham’s F-12 (Gibco, #11330-032), supplemented with 5% Horse Serum, (Gibco, #16050-122), 20 ng/mL epidermal growth factor (Peprotech, #AF-100-15), 0.5 µg/mL hydrocortisone (Sigma, #H-0888), 100 ng/mL cholera toxin (Sigma, #C-8052), 10 µg/mL insulin (Sigma, #I-1882), and 1% penicillin-streptomycin (Gibco, #15240-062).A total of approximately 50 million cells were used for each experimental relicate.
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| Extracted molecule |
other |
| Extraction protocol |
Total RNA was extracted (Quick RNA, Zymo, #R1055). 30 µg per sample of poly-adenylated mRNA was isolated using oligo d(T) beads (NEB E7490L).
Resulting mRNA was split into 6 separate reactions and cDNA was synthesized using a gene-specific primer (SL1972) and MultiScribe Reverse Transcriptase (Invitrogen, 4311235) . Following cDNA synthesis, all cDNA samples were pooled and one junction-PCR reaction was performed with 16 cycles (SL1973, SL1974) and I5 and i7 primers from NEBNext Oligo Kit (E7600S) were used for Illumina barcoding with between 5 and 9 cycles. MPRA plasmid DNA pools were amplified with i5 and i7 primers as a control for oligo representation. All libraries were pooled and sequenced as single-end, 100bp on an Illumina NextSeq 2000 instrument at the University at Albany Center for Functional Genomics.
STARR-Seq
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
PCR amplified plasmid DNA (STARR-seq DNA)
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| Data processing |
Raw reads were counted by searching for exact match in the designed enhancer library pool sequences in the fastq file using a custom python script.
Assembly: Assembly: GRCh38
Supplementary files format and content: Comma-delimited, raw read counts for each enhancer variant sequence in plasmid DNA pool and STARRSeq RNA from each condition.
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| Submission date |
May 06, 2024 |
| Last update date |
May 08, 2024 |
| Contact name |
Morgan A Sammons |
| E-mail(s) |
[email protected]
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| Organization name |
University at Albany - SUNY
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| Department |
Biological Sciences
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| Lab |
Sammons
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| Street address |
1400 Washington Ave, Life Sciences 2075
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| City |
Albany |
| State/province |
NY |
| ZIP/Postal code |
12222 |
| Country |
USA |
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| Platform ID |
GPL32628 |
| Series (1) |
| GSE266670 |
Context dependent activity of p63-bound gene regulatory elements |
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| Relations |
| BioSample |
SAMN41240450 |
| SRA |
SRX24476307 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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