|
Status |
Public on Sep 20, 2017 |
Title |
0B |
Sample type |
SRA |
|
|
Source name |
HUH7 hepatoma cell line
|
Organism |
Homo sapiens |
Characteristics |
cell line: HUH7 time point: 0
|
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).
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Description |
EU-RNA replicate B, labeled with EU at 0 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.
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|
|
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 |
SAMN05851534 |
SRA |
SRX2198985 |