|
| Status |
Public on Sep 20, 2017 |
| Title |
105C |
| Sample type |
SRA |
| |
|
| Source name |
HUH7 hepatoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HUH7
time point: 105
|
| Treatment protocol |
Cells were pulse labeled with 0.5 mM EU in standard growth media for 40 minutes at 37˚C
|
| Growth protocol |
HUH7 human hepatoma cells were cultured in DMEM High Glucose with L-glutamine (Invitrogen, 11965), 10% FBS (HyClone), and 1% penicillin-streptomycin (Invitrogen) at 37˚C in 5% CO2. For mitotic arrest experiments, the cells were synchronized at the G1/S transition by culturing for 24 hr in complete DMEM supplemented with 2 mM thymidine (Sigma, T1895). The thymidine media was removed and cells were washed once with PBS. The cells were then cultured for 2 hr in complete DMEM to complete synchronization. The media was then supplemented with nocodazole (Sigma, M1404) for a final concentration of 0.06 ug/mL, and incubated at 37˚C for 16 hours. Mitotic cells were manually collected by shake-off. To release the cells from arrest, mitotic cells were washed twice with PBS and re-plated in complete DMEM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The media was removed and the cells were collected with TRIzol Reagent (Ambion). Total RNA was isolated using miRNeasy (Qiagen). Biotin-azide was then tethered to EU with the Click-iT Nascent RNA Capture Kit (Invitrogen). Biotin-EU-RNAs were pulled down from total RNA using streptavidin-coated magnetic beads (Invitrogen).
The streptavidin bead-tethered biotin-EU-RNA was used for stranded, ribo-depleted cDNA library generation (Ovation Human FFPE RNA-seq Multiplex System).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
EU-RNA
replicate C, labeled with EU at 105 minutes of release from nocodazole block
|
| Data processing |
sequenced fragments were demultiplexed with bcl2fastq
fragments were trimmed to 35 nucleotides
fragments were aligned with Bowtie2 2.2.9, using the tophat "very sensitive" option in end-to-end mode
aligned BAM files were converted to BED files with bamToBed
BED files were reduced to unique, non-PCR duplicated fragments
fragments were extended to a total length of 300 nucleotides
BED files were normalized to the genome-wide RPM to make bedGraphs
NoEU signal was subtracted on a per base pair basis from each experimental sample
negative values were removed from all NoEU-subtracted samples
FPKM for every hg19 RefSeq was measured with a custom script, available upon request
converted bedGraphs to bigWigs with bedGraphToBigWig
Genome_build: hg19
Supplementary_files_format_and_content: bigWig UCSC Genome Browser tracks on sample records, FKPM file on series record.
|
| |
|
| Submission date |
Sep 29, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Katherine Claire Palozola |
| E-mail(s) |
palozola@mail.med.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Cell and Developmental Biology
|
| Lab |
Kenneth S. Zaret
|
| Street address |
3400 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE87476 |
Mitotic Transcription and Waves of Gene Reactivation During Mitotic Exit |
|
| Relations |
| BioSample |
SAMN05851527 |
| SRA |
SRX2198995 |
