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| Status |
Public on Jun 29, 2020 |
| Title |
Ancestral FAS exon 6 Library Output 8c |
| Sample type |
SRA |
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| Source name |
Cell culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
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| Growth protocol |
For each experimental replicate, 10 ng of the library were transfected into 250 000 HEK293 cells in one well of a 6-well plate using Lipofectamine 2000 (11668027, Thermo scientific) and OPTIMEM I Reduced Serum Medium (31985-047, Thermo scientific). Six hours post-transfection, the cell culture medium was replaced with DMEM Glutamax (61965059, Life Technologies) containing 10% FBS and Pen/Strep antibiotics. 48 hours post-transfection, total RNA was isolated using the automated Maxwell LEV 16 simplyRNA tissue kit (AS1280, Promega).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each of the experimental replicates, 10 ng of the library were transfected into 250 000 HEK293 cells in one well of a 6-well plate using Lipofectamine 2000 (11668027, Thermo scientific) and OPTIMEM I Reduced Serum Medium (31985-047, Thermo scientific). Six hours post-transfection, the cell culture medium was replaced with DMEM Glutamax (61965059, Life Technologies) containing 10% FBS and Pen/Strep antibiotics. 48 hours post-transfection (72 in the case of the siRNA experiments), total RNA was isolated using the automated Maxwell LEV 16 simplyRNA tissue kit (AS1280, Promega).
Accuprime Pfx (12344024, ThermoFisher Scientific) was used following the manufacturer's instructions to amplify 20 ng of single-stranded library DNA for 25 cycles with the following flanking intronic primers: FAS_i5_GC_F and FAS_i6_GC_R (Baeza-Centurion et al. 2019). The amplified library was then recombined with pCMV FAS wt minigene exon 5-6-7 (Förch et al. 2000) using the In-Fusion HD Cloning kit (639649, Clontech) in a 1:8 vector:insert optimized ratio and transformed into Stellar competent cells (636766, Clontech) to maximise the number of individual transformants (800,000 individual clones). The library was then amplified by growing for 18 hours in LB medium containing ampicillin. The final plasmid library were purified using the Quiagen plasmid maxi kit (50912163, Quiagen) and quantified with a NanoDrop spectrophotometer.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
PCR amplicon
Mature mRNA containing the ancestral FAS exon 6.
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| Data processing |
Library strategy: (DNA-seq of PCR amplicon)
Demultiplex sample replicates (DiMSum - https://github.com/lehner-lab/DiMSum)
Paired-end merging (DiMSum - https://github.com/lehner-lab/DiMSum)
Base-calling (DiMSum - https://github.com/lehner-lab/DiMSum)
Supplementary_files_format_and_content: DiMSum output file (tab-delimited plain text file)
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| Submission date |
Jun 06, 2020 |
| Last update date |
Jun 29, 2020 |
| Contact name |
Pablo Baeza |
| Organization name |
Centre for Genomic Regulation
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| Department |
Systems Biology
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| Lab |
Genetic Systems
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| Street address |
Dr. Aiguader, 88
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| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE151942 |
Mutations primarily alter the inclusion of alternatively spliced exons |
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| Relations |
| BioSample |
SAMN15154428 |
| SRA |
SRX8485182 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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