|
| Status |
Public on Dec 31, 2020 |
| Title |
Experiment_02_g2G3 |
| Sample type |
SRA |
| |
|
| Source name |
human cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Hela cells
genotype: XRCC4(-/-)
treatment: XRCC4 knockout
experiment: Experiment_02
|
| Treatment protocol |
To create a XRCC4(-/-) cell line to block cNHEJ, a gXRCC4 sequence targeting the XRCC4 gene (NCBI RefSeq: NC_000005.10) was annealed, phosphorylated with T4 Polynucleotide Kinase (New England Biolabs) and cloned into the BsmBI digested LenticrisprV2 plasmid (Addgene). The plasmid was delivered into HeLa cells by lentiviral transduction.
|
| Growth protocol |
HeLa (ATCC® CCL-2™) cells obtained from the ATCC and LentiX293T (Clonetech) were grown in Dulbecco modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (HyClone). To assess DNA repair, HeLa cells (0.6 million) were seeded and after one day, co-transfected with a NHEJ reporter plasmid with either pairs of TALEN reporter constructs. Media was changed after 6 hours and cultured in complete media (DMEM+ 10% FBS), with or without mirin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells post 48 hours transfection were harvested and RNA was extracted using Zymogen RNA prep kit (Zymogen).
The RNA sample library preparation and sequencing on the Illumina platform PE150 was performed at Novagene Corporation Inc.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Hela cells with only blocking C-NHEJ pathway by XRCC4 knockout in Experiment_02
all_sample_human_RNA_max.txt
|
| Data processing |
Sequenced reads were imported into CLC Genomics Workbench 12.0 (https://www.qiagenbioinformatics.com/) and trimmed for adaptor sequence using quality limit of 0.05 calculated from a modified-Mott trimming algorithm, read through adapter trimming, and trimming of ambiguous bases from read regions with more than 2 ambiguous reads.
RNA seq analysis was performed with the RNA Seq Analysis tool in Genomics Workbench which is based upon the ENRAGE pipeline2. Default settings were used including a mismatch cost of 2, insertion and deletion cost of 3.
Read 1 and read 2 from pairs could be processed independently as technical replicates.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include read counts for each raw read file
|
| |
|
| Submission date |
Aug 01, 2019 |
| Last update date |
Dec 31, 2020 |
| Contact name |
Martin Schiller |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Nevada, Las Vegas
|
| Department |
Nevada Institute of Personalized Medicine
|
| Street address |
4505 S Maryland Pkwy
|
| City |
Las Vegas |
| State/province |
NV |
| ZIP/Postal code |
89154 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE135274 |
Transcriptome changes of Hela cells with blocking classical and alternative non-homologous end-joining (NHEJ) pathways |
|
| Relations |
| BioSample |
SAMN12423344 |
| SRA |
SRX6639247 |
