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| Status |
Public on Jul 01, 2024 |
| Title |
Mutagenesis_screen_bin8_r2_RNA |
| Sample type |
SRA |
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| Source name |
Replicate 2, Bin 8, Mutagenesis screen
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
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| Treatment protocol |
Cells were infected with lentivirus containing a plasmid library. Post-infection, cells underwent puromycin selection until recovery with >90% viability. The infection rate was kept below 30% as estimated by the fraction of mCherry-positive cells.
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| Growth protocol |
The HEK293 cells (ATCC CRL-3216) were cultured in DMEM high-glucose medium supplemented with 10% FBS, L-glutamine (4 mM), sodium pyruvate (1 mM), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (1 μg/mL) (Gibco).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following FACS sorting of cells based on eGFP/mCherry expression, genomic DNA was extracted from pelleted cells using the NucleoSpin Blood kit from Macherey Nagel, chosen based on cell quantity.
Libraries were constructed using Illumina-compatible primers designed for Addgene #213963 plasmid, incorporating staggered regions to ensure cluster diversity. PCR amplification was performed using NEBNext Ultra II Q5 Master Mix, followed by product purification with the Select-a-Size DNA Clean & Concentrator MagBead Kit (Zymo Research). Quality was verified using Agilent TapeStation or BioAnalyzer.
The libraries were sequenced to achieve a depth of >20 million reads per sample, with each sample uniquely barcoded using a combination of i5 and i7 indexes. This setup ensures sufficient coverage and identification of each library component in the pooled sample.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
mutagenesis_screen.tsv
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| Data processing |
Adaptor Removal: Used cutadapt to trim adapter sequences specific to vector-based primers.
Reference Preparation: Created and indexed a reference sequence database of oligonucleotides. Generated a GTF annotation from oligonucleotide sequences.
Mapping: Aligned reads to the reference using bwa mem, ensuring at least 80% of reads aligned exactly once. Sorted and indexed aligned reads using samtools.
Read Counting, Normalization and Analysis: Counted reads aligning to genes with featureCounts, followed by replication correlation assessment. Normalized read counts using median of ratios method to correct for library size variations. Calculated correlations between read counts across sorting bins to rank candidate RNA switches.
Assembly: The analysis does not involve a standard genome build but utilizes a custom reference composed of synthesized oligonucleotide sequences tailored to the experimental design.
Supplementary files format and content: Tab-separated files with raw read counts for each oligonucleotide.
Library strategy: MPRA
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| Submission date |
Apr 28, 2024 |
| Last update date |
Jul 01, 2024 |
| Contact name |
Hani Goodarzi |
| Organization name |
UCSF
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| Department |
Biochemistry and Biophysics
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| Street address |
600 16th St, GH S312D
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE266058 |
Massively parallel reporter assay for identification of RNA switches |
| GSE266070 |
RORC RNA Switch Mechanisms |
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| Relations |
| BioSample |
SAMN41106000 |
| SRA |
SRX24387227 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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