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| Status |
Public on Oct 28, 2016 |
| Title |
WT hepatocyte rep3 |
| Sample type |
SRA |
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| Source name |
hepatocyte
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| Organism |
Mus musculus |
| Characteristics |
strain: B6.129S6-Myctm2Fwa
tissue: Liver
cell type: hepatocyte
genotype: WT
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using Qiagen RNeasy columns (Valencia, CA). Each RNA sample was assessed for quality and quantity using a Qubit 2.0 fluorometer and Agilent Bioanalyzer TapeStation 2200. The Sample preparation was replicated using Illumina TruSeq Stranded mRNA kit (San Diego, CA). mRNA was used to generate poly-A+ libraries using oligo-dT beads following 2 rounds of poly-A selection.
mRNA was fragmented to an average length of 260 nt by magnesium-catalyzed hydrolysis at 940C and then converted into cDNA by random priming. cDNA 3’ ends were adenylated, followed by adaptor ligation and a 9-cycle PCR to enrich DNA fragments. cDNA libraries were then validated using KAPA Biosystems (Wilmington, MA) primer premix kit with Illumina-compatible DNA primers on the Qubit 2.0 fluorometer and Tapestation 2200 systems described above. Libraries were diluted in 0.1% Tween 20 to concentrations of 2 nM for stabilization. The cDNA libraries were then pooled to final concentrations of 2.5 pM.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
1b-130-WT
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| Data processing |
Raw sequencing reads were quality checked for potential sequencing issues and contaminants using FastQC (Babraham Bioinformatics, Inc, Cambridge, UK.). Adapter sequences, primers, Ns, and reads with quality score below 28 were trimmed using fastq-mcf of ea-utils and PRINSEQ (http://prinseq.sourceforge. net/manual.html). Reads with a remaining length of less than 20 bp after trimming were discarded. Paired end reads were mapped to the mouse genome (m10) using TopHat (http://ccb.jhu.edu/software/tophat/index.shtml) in a strand specific manner. Read coverage on forward and reverse strands for genome browser visualization was computed using SAMtools, BEDtools, and UCSC Genome Browser utilities. Pairwise differential expression was quantified using Cuffdiff and DESeq [4, 5]. Cufflinks was used to determine FPKM levels for each gene from the TopHat alignment and was used as input for Cuffdiff. Significant differentially expressed genes were determined by adjusted P-value with a threshold of 0.05. DESeq was utilized
for raw read counts, which were calculated by HTSeq based on the TopHat alignment (http://www-huber.embl.de/users /anders/HTSeq/doc/count.html). Read counts were then normalized across all samples and significant differentially expressed genes were determined by adjusted P-value with a threshold of 0.05
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
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| Submission date |
Oct 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Edward V Prochownik |
| E-mail(s) |
edward.prochownik@chp.edu
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| Phone |
412-692-6795
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| Organization name |
Children’s Hospital of Pittsburgh of UPMC
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| Department |
Division of Hematology/Oncology
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| Street address |
4401 Penn Ave.
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15224 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE87578 |
Coordinated Activities of Multiple Myc-Dependent and Myc-Independent Biosynthetic Pathways in Hepatoblastoma |
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| Relations |
| BioSample |
SAMN05859812 |
| SRA |
SRX2208277 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2334711_WT_Hepa_replicate3_cufflinks_genes.fpkm_tracking.gz |
692.3 Kb |
(ftp)(http) |
FPKM_TRACKING |
| GSM2334711_WT_Hepa_replicate3_cufflinks_isoforms.fpkm_tracking.gz |
1014.8 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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