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| Status |
Public on Jun 15, 2015 |
| Title |
H2C |
| Sample type |
SRA |
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| Source name |
HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Epithelial fetal human kidney cells
cell modifications: Contains SV40 T-antigen
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| Growth protocol |
Both cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 2mM Penicillin-Streptomycin (Invitrogen), at 37°C in 5% CO2. The cells were allowed to reach 80-90% confluence before harvesting with trypsin-EDTA.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from HEK 293T and IMR-90 cells using TRIzol extraction. Four biological replicates from each cell line were DNase treated, and Ribo-Zero rRNA removal (Ribo-Zero, Epicentre) was utilized for three of the four RNA samples, leaving a non-Ribo-Zero depleted sample for rRNA expression analysis.
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. The samples were multiplexed and sequenced using 100 bp single-end read sequencing on the Illumina HiSeq 2500 in our institutional Epigenomics Shared Facility.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
DNase-treated, rRNA-depleted mRNA
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| Data processing |
After sequencing, fastq file generation was completed using the Illumina CASAVA pipeline (v1.8).
Post-sequencing analysis was performed using the WASP pipeline (v3.1.5 rev. 6632), involving read alignment using gsnap (2012-07-20), with htseq (v0.5.3p3) used to determine read quantitation.
Biological replicates were normalized using DESeq (Bioconductor) and RefSeq gene identifiers were assigned using biomaRt.
Only gene expression assigned a RefSeq identifier was used for further analysis.
Genome_build: hg19
Supplementary_files_format_and_content: bed file - containing gene chr, start, stop, refseq id, ensemble id, expression, strand, and expression quartile
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| Submission date |
May 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie Nadel |
| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Lab |
Price 314
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| Street address |
1301 Morris Park Avenue
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE68938 |
RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships (RNA-seq) |
| GSE68953 |
RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships |
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| Relations |
| BioSample |
SAMN03658789 |
| SRA |
SRX1029432 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1687389_H2C.txt.gz |
200.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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