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| Status |
Public on Jul 15, 2019 |
| Title |
L16_1137_Rat_3_Pituitary |
| Sample type |
SRA |
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| Source name |
Pituitary
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| Organism |
Rattus norvegicus |
| Characteristics |
organism common name: Rat
strain: Brown Norway
individual: 3
age: 9 weeks
rna integry number (rin): 9.7
library prep batch: 2
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| Treatment protocol |
RNALater (Ambion)
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| Growth protocol |
standard lab/breeding colony conditions
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tissues were isolated from freshly sacrificed animals (< 1 hour from time of death to tissue fixation), washed in cold PBS, and stored in RNALater (Ambion) per the manufacturer’s instructions. Each sample was then homogenized in a bead-mill in Trizol and total RNA was isolated by and treated with DNAse on a Qiagen miRNEasy column
Libraries were prepared using the TruSeq mRNA library kit (Illumina) according to manufacturer’s instructions with the following modifications: After prep, libraries were size-selected using 2% agarose gels on the PippinHT system (Sage Science) with a capture window of 300-600 bases. The size-selected material was then subjected to one additional cycle of PCR with fresh reagents and P5/P7 primers (Illumina) to ensure all library fragments were fully double-stranded. This step significantly reduced the percentage of smaller fragments co-purifying in the final gel size-selection. After PCR, libraries were size-selected a second time on the PippinHT with the same settings
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
rat.salmon.tximport.counts.txt
rat.salmon.tximport.tpm.txt
rat.stringtie.gtf
rat.tx2geneid.txt
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| Data processing |
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500
Libraries from the non-human mammals were mapped to their respective reference genomes using STAR v2.5.0 with the following parameters: --outFilterMultimapNmax 50 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.15
StringTie v1.3.3b with the --rf parameter was run on each resulting .bam file to assemble novel isoforms relative to the reference transcriptome annotation
The sets of novel and existing transcript annotations from each library were then compiled into a single set of annotations for each species using the --merge option in StringTie v1.3.3b
Abundances for these sets of transcripts for each species were then quantified using salmon v0.9.1 with the following parameters/flags: --seqBias, --gcBias, --useVBOpt
The tximport R package (with countsFromAbundance = “lengthScaledTPM”) was used to sum transcript-level counts and transcripts per million (TPM) values from salmon to the gene level for all transcripts with the following class codes: c, j, e, o, =, u
Genome_build: cyno, macFas5; dog, canFam3; mouse, mm10; rat, rn6
Supplementary_files_format_and_content: Transcript annotations for each species are provided as .gtf files. Assignments of transcripts (Ensembl ID for known, Stringtie ID for novel) to genes (Ensembl ID) along with transcript class codes are provided as tab-delimited .txt files. Gene-level TPM and counts are provided as tab-delimited .txt files
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| Submission date |
Jan 22, 2019 |
| Last update date |
Jul 15, 2019 |
| Contact name |
Sahin Naqvi |
| E-mail(s) |
sahin.naqvi@gmail.com
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| Organization name |
Whitehead Institute
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| Lab |
Page
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| Street address |
455 Main St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE125483 |
Conservation, acquisition, and functional impact of sex-biased gene expression in mammalian tissues |
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| Relations |
| BioSample |
SAMN10789794 |
| SRA |
SRX5279695 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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