|
| Status |
Public on Nov 14, 2024 |
| Title |
HeLa cells, Arsenite, XRN1 siRNA, 60 min, rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
HeLa cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa cell line
genotype: WT
treatment: Arsenite, XRN1 siRNA
time (arsenite_treatment): 60 minute
kit: SQK-RNA002
flow-cell: FLO-PRO002
|
| Treatment protocol |
For arsenite treatment, 80-85% confluent HeLa cells were treated with 500 µM of arsenite (Sigma) whereas for combined arsenite and cycloheximide treatment, cells were treated with 500 µM of HeLa cells and 25 µg/ml of cycloheximide for 1 hour. Similarly for combined arsenite and ISRIB treatment, cells were treated with 500 µM of arsenite and 200 nM of ISRIB for 1 hour.
For siRNA treatment, HeLa cells were transfected with 20 nM of siRNA specific to XRN1 using Neon Transfection system 100 µl kit (ThermoFisher, MPK100) for 72 hours.
|
| Growth protocol |
HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37C
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA from HeLa cells was isolated using Trizol reaget (Invitrogen, Cat# 15596-018) following the manufacturer’s protocl. 75 µg of total RNA was used to isolate poly(A) mRNAs. 500-1000 ng of poly(A) mRNA was used for library preparation
RNA libraries for dRNA-Seq were prepared using direct RNA sequencing kit (Oxford Nanopore Technologies, SQK-RNA002) following manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Data processing |
Basecalled using Guppy (v3.4.5)
5’ adaptors were identified and removed using Cutadapt (v2.8)
Reads were first aligned against ribosomal sequences obtained from SILVA. Non-ribosomal reads were subsequently mapped against the human genome hg38 using minimap2 (version 2.17)
Reads were also aligned against the human transcriptome.
The poly(A) tail lengths were extracted from sequenced reads using the nanopolish polya package, with quality control scores as “PASS” selectively used in our analysis.
Assembly: hg38
Supplementary files format and content: *counts_length.tab: Tab-delimited text files include transcript id based raw counts and respective average trasncript length values for each Sample
Supplementary files format and content: *polyA.tab: PolyA length
Supplementary files format and content: DESeq2 differential gene expression (Arsenite-Control)
|
| |
|
| Submission date |
May 25, 2022 |
| Last update date |
Nov 14, 2024 |
| Contact name |
Emmanouil Maragkakis |
| E-mail(s) |
[email protected]
|
| Phone |
+1 410-454-8429
|
| Organization name |
National Institute on aging
|
| Department |
Laboratory of Genetics and Genomics (LGG)
|
| Lab |
Computational Genomics Unit
|
| Street address |
251 Bayview Blvd, 10B133 Baltimore
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL26167 |
| Series (1) |
| GSE204785 |
Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress |
|
| Relations |
| BioSample |
SAMN28657257 |
| SRA |
SRX15450690 |
