|
| Status |
Public on Feb 13, 2015 |
| Title |
1.0_Recovery |
| Sample type |
SRA |
| |
|
| Source name |
HEK 293T cells, cells allowed to recover for one month after the 5-aza-CdR treatment
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293T
cell type: non-malignant cell line
|
| Treatment protocol |
HEK 293T cells were treated for three days with 1.0 µM of 5-aza-CdR. Cells with no drug were grown as controls. Tissue culture medium was changed every day for both control and treated cells, to maintain the drug stability during treatment. After collection, treated cells were remained in culture for a further 30 days.
|
| Growth protocol |
HEK 293T cells were grown in triplicates, in DMEM supplemented with 10% fetal bovine serum and 2 mM Penicillin-Streptomycin at 37°C in 5% CO2. Cells were passaged only when reaching 80-90% confluence, for a total period of one month.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
HEK 293T cells were collected by washing and trypsinizing for three minutes. The same procedure was performed with cells allowed to remain in culture for 30 days without the drug. Total RNA was extracted with Trizol (Invitrogen) and rRNA depleted with Ribozero (Epicentre).
cDNA synthesis was performed by using oligo-dT as well as random hexamers. Actinomycin D was added to the reaction to prevent any possible amplification from contaminating genomic DNA. During second strand synthesis, a dU/VTP mix was used to create directional libraries. Before library preparation, cDNA samples were Covaris-fragmented to 300 bp fragments. The samples were then end-filled, 3’ terminal A-extended and ligated to pre-annealed TruSeq indexed Illumina adapters. Uracil-DNA-glycosylase (UDG) treatment preceded the PCR reaction to cleave the uridine-containing strand and therefore identify the orientation of each transcript
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
DNase-treated, rRNA-depleted mRNA
|
| Data processing |
sequencing reads were aligned by the WASP pipeline version 3.1.3 (rev. 6589), using gsnap (2012-07-20)
FASTQ files incorperate Phred quality scores. NOTE: In the raw sequence data fastq file reads failing Illumina's purity filter are removed automatically
Cuffflinks was performed for each sample to calculate FPKM values, specifying the strand of the reads (fr-fisrtstrand), performing an initial estimation of the number of reads mapping in multiple locations in the genome (-u), and normalizing by the upper quartile of the number of fragments mapping to individual loci (-N). To calculate differences in expression between samples, the cufflinks results were merged by cuffmerge using a mm9 GTF file obtained from the UCSC Genome Browser as a reference genome, and then used by cuffdiff to calculate differentially expressed genes
Genome_build: hg19
Supplementary_files_format_and_content: tables containing all RefSeq and lncRNA Genes, analyzed by Cufflinks (Cuffdiff) in order to calculate FKPM values.
|
| |
|
| Submission date |
Oct 22, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Maria-Paz Ramos |
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Genetics
|
| Lab |
John Greally
|
| Street address |
1301 Morris Park Ave, Price 314
|
| City |
Bronx |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE62590 |
DNA demethylation by 5-aza-2'-deoxycytidine is imprinted targeted to euchromatin and has limited transcriptional consequences |
| GSE62591 |
HEK 293T cells RNA-seq data treated with 5-aza-2-deoxycytidine |
|
| Relations |
| BioSample |
SAMN03135177 |
| SRA |
SRX738251 |
