|
| Status |
Public on Jan 02, 2023 |
| Title |
MPRE_input_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: human, mouse, yeast, synthetic
cell line: HEK293T
cell type: human embryonic kidney
treatment: MPRE plasmids transfection, MPRE contains ~12000 mouse, human, yeast and synthetic sequences
|
| Treatment protocol |
0.5 million HEK293T cells were seeded in each well of a 6-well plate. 24 hours later cells were transfected with 2 μg plasmid DNA and 8 ul home-made PEI reagent. Cells were harvested 24 hours post transfection. Each 3 wells of the 6-well plate were merged to form two replicates.
|
| Growth protocol |
HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% Penicillin and Streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with NucleoZOL (Macherey-Nagel)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech). After mild fragmentation, samples were divided into input and IP sample, and IP samples were subjected to m6A immunoprecipitation. RNA from both the input and IP samples was reverse transcribed with a sequence specific RT primer to include the 10 nt unique barcode sequence of each construct in the MPRE. PCR amplicons of these fragments were generated.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep1
|
| Data processing |
Fastq files were processed with a custom R script. Barcodes matching the planned barcode were counted for each sample. See Table S2 for the planned barcode sequences.
MPRE sequences based on mm9 and hg19, no alignment was performed
Csv file (BCcounts_raw.csv) containing raw barcode counts of each construct in the MPRE for input and IP of both biological relicates. Excel file (TableS2.xlsx) containing all planned construct sequences and description.
|
| |
|
| Submission date |
May 27, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Anna Uzonyi |
| E-mail(s) |
uzonyianna@gmail.com
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Department of Molecular Genetics
|
| Lab |
Schraga Schwartz
|
| Street address |
234 Herzl street
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE204980 |
Exclusion of m6A from splice-site-proximal regions by the exon-junction complex dictates m6A topologies and mRNA stability |
|
| Relations |
| BioSample |
SAMN28697727 |
| SRA |
SRX15483201 |
