|
| Status |
Public on Dec 01, 2023 |
| Title |
HepG2, tunicamycin, rep 5 |
| Sample type |
SRA |
| |
|
| Source name |
HepG2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
library type: MPRA RNA
treatment: tunicamycin
replicate: e
library: HepG2-ATF4MPRA_Tunicamycin_rep2e
|
| Treatment protocol |
9 h after transfection, the medium was replaced with regular growth medium (described above) with or without 2 ug/ml tunicamycin, and the cells were harvested 17 h later. To generate replicates, the transfection and treatment of cells were repeated on separate days (n=5).
|
| Growth protocol |
HepG2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; 4.5 g/l glucose; Gibco) supplemented with 10% fetal bovine serum and 1x penicillin-streptomycin. 6×10e6 cells per 10 cm plate were transfected with 22 ug MPRA plasmid library using the Lipofectamine 3000 transfection reagent (Thermo Scientific) and Opti-MEM I reduced-serum medium (Thermo Scientific).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from the cells using the RNeasy Midi Kit (Qiagen) according to the procedure described by the manufacturer. Poly(A)+ RNA was subsequently isolated using Dynabeads Oligo(dT)25 beads (Invitrogen), treated with DNase I (Thermo Scientific) and purified using the RNeasy Micro kit (Qiagen).
Poly(A)+ RNA was reverse-transcribed with SuperScript III (Invitrogen) using primer specific to the MPRA reporter RNA. The RT primer incorporated a 12-nt UMI (unique molecular identifier). To generate the MPRA plasmid library 'input' NGS libraries, the MPRA plasmid library was used as template in an DNA synthesis reaction with Phusion Hot Start II High-Fidelity DNA Polymerase. The resulting products were used to prepare targeted (amplicon) NGS libraries to quantify the MPRA barcodes.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
MPRA RNA readout
|
| Data processing |
Reads were trimmed to retain 35 bp from the start of Read1 (expected to contain the 10-bp MPRA sequence barcode followed by 25 bp of constant flanking sequence). The reads were then mapped using Bowtie (version 1.3.0) to a synthetic genome consisting of 35 bp contigs (each of the possible 10-bp barcodes attached to 25 bp of constant sequence). In mapping, one nucleotide mismatch was allowed and any multimapping reads were discarded.
After mapping, the UMI read (12bp, the entire Read2) was used for UMI deduplication with UMI-tools (version 1.1.0) to filter out likely PCR duplicate reads.
After UMI deduplication, featureCounts from the Subread package was used to generate the raw count matrix.
Assembly: hg38
Supplementary files format and content: Tab-separated text file containing raw counts for all MPRA oligonucleotides (barcodes) in all libraries. Extra columns provide metadata for each MPRA oligo, such as the genetic variant position in the hg38 genome and the alleles tested.
Library strategy: MPRA RNA barcode amplicon
|
| |
|
| Submission date |
Feb 13, 2023 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Tiit Örd |
| E-mail(s) |
tiit.ord@ut.ee
|
| Organization name |
University of Tartu
|
| Department |
Institute of Genomics
|
| Street address |
Riia 23b/2
|
| City |
Tartu |
| ZIP/Postal code |
51010 |
| Country |
Estonia |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE225216 |
High-throughput reporter assay for human genetic variants overlapping ATF4 binding sites |
|
| Relations |
| BioSample |
SAMN33272761 |
| SRA |
SRX19355137 |
