|
| Status |
Public on Mar 01, 2019 |
| Title |
P14.hep.Wt.sexM.bio1 (RNA-Seq) |
| Sample type |
SRA |
| |
|
| Source name |
P14.hep.Wt.sexM
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wt
age: postnatal day 14
Sex: male
cell type: hepatocytes
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Hepatocytes were resuspended in TRIzol (Thermo Fischer 15596026) and stored at -20°C until the day of library preparation, when RNA was isolated from TRIzol per the manufacturer’s protocol and brought through at least cDNA synthesis all on the same day. RNA isolated from TRIzol was resuspended in TE. Next, total RNA was polyA-selected using 50 μL Invitrogen Dynabeads (dT)25-61002 per the manufacturer’s protocol. Sequencing libraries were prepped from polyA+ RNA using the NEBNext Ultra Directional Library Prep Kit for Illumina (NEB 7420), with 12 cycles of PCR using NEBNext Oligos. After library prep, library size was assessed using a Bioanalyzer and Agilent DNA 1000 chip (Agilent 5067-1504), and the concentration of amplifiable DNA assessed with the NEBNext Library Quantification Kit (NEB E7630). Individual libraries were diluted to 2 nM, pooled, and the pooled library requantified with the NEBNext Next Library Quantification Kit. The pooled library was diluted to 3.2 pM using the NEBNext 500/550 High Output Reagent Cartridge V2 75 cycles (Ref 15057934) and loaded onto the NextSeq 500 for 75bp single-end sequencing.
NEBNext Ultra Directional Library Prep Kit for Illumina (NEB 7420)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
rawCounts.txt
normCounts.txt
P14.hep.Wt.sexM.Grindheim.SS.2462.1
|
| Data processing |
Merging: Sequencing replicates were concatenated before alignment.
Alignment: Reads were aligned to NCBI v37/mm9 build from the UCSC Genome Browser with STAR 2.4.2a with the following parameters: ----outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000
Quantitation: Alignments were assigned to mm9 RefSeq (refFlat, from UCSC genome table browser) transcripts with HTSeq v0.6.1p1 and the following arguments: -f sam -r pos -t exon -i gene_id -s no
Differential testing: The count table from HTSeq was normalized and differential gene expression called with DESeq2 v1.14.1
Track creation: Alignments greater than 75bp (corresponding to splicing events) were filtered out to avoid spurious intronic signal. Reads were split into plus and minus strands, RPM-normalized bedgraphs were generated with genomeCoverageBed (UCSC utilities), and converted to bigWigs with bedgraphToBigWig (UCSC utilities).
Genome_build: mm9
Supplementary_files_format_and_content: BigWigs represent plus and minus strand RNA signal. TXT files represent raw (HTseq output) and normalized (DESeq2 output) counts.
|
| |
|
| Submission date |
Aug 20, 2018 |
| Last update date |
Mar 01, 2019 |
| Contact name |
Jessica Mae Grindheim |
| E-mail(s) |
jmgrindheim@gmail.com
|
| Organization name |
UNIVERSITY OF PENNSYLVANIA
|
| Department |
Cell and Developmental Biology
|
| Lab |
Kenneth S Zaret
|
| Street address |
3400 Civic Center Blvd SCTR 9-169
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19146 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE118757 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation [RNA-Seq] |
| GSE119219 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation |
|
| Relations |
| BioSample |
SAMN09862801 |
| SRA |
SRX4578062 |
