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Sample GSM2332399 Query DataSets for GSM2332399
Status Public on Sep 20, 2017
Title 80B
Sample type SRA
Source name HUH7 hepatoma cell line
Organism Homo sapiens
Characteristics cell line: HUH7
time point: 80
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 B, labeled with EU at 80 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
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
BioSample SAMN05851531
SRA SRX2198991

Supplementary file Size Download File type/resource 450.6 Mb (ftp)(http) BW 406.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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