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| Status |
Public on Nov 21, 2018 |
| Title |
Anterior, 6 dpb, replicate 2 |
| Sample type |
SRA |
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| Source name |
Ant, 6d
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| Organism |
Patiria miniata |
| Characteristics |
segment: regenerating anterior segments
time point: 6 dpb
replicate: 2
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| Treatment protocol |
Patiria miniata larvae (7 days post fertilization) were manually bisected stereotypically through the foregut, midway along the transverse anterior-posterior axis with a #11 sterile scalpel. Resulting anterior and posterior segments, as well as sibling control (non-bisected) larvae, were then transferred to separate 35 mm polystyrene dishes at a density of no more than 50 larval segments per ml of artificial seawater. Pools of of approximately 300 sibling regenerating posterior segments, anterior segments, and non-bisected sibling control larvae were collected at three points following bisection: one early (approximately 3 hours post bisection, hereafter 0 dpb), one intermediate timepoint (3 dpb), and one timepoint at the initiation of blastemal proliferation (6 dpb). Two biological replicate samples were prepared for each timepoint.
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| Growth protocol |
Regenerating and control larvae were cultured in artificial seawater at 16 °C and fed Rhodomonas les ad libitum every 2 days along with fresh artificial seawater beginning at 4 days post fertilization (dpf).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using the GenElute Mammalian Total RNA Kit (Sigma-Aldrich).
RNA libraries were prepared for sequencing using standard Illumina protocols (TruSeq)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Basecalls performed using CASAVA version 1.8
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Pmin_1.0 draft genome assembly using tophat v2.0.12 with parameters: --no-coverage-search --library-type fr-unstranded --b2-very-sensitive --transcriptome-index <bwtidx> -G <pm.genes.gff3>. Alignments were filtered to include only uniquely mapped reads using samtools 0.1.19-44428cd. Transcripts were assembled using cufflinks v2.2.1 with parameters: -u -b <pmin.scaff.fa> -g <pm.genes.gff3> and all cufflinks assemblies were merged using cuffmerge.
Transcript abundance was calculated against the merged transcriptome assemblies using HTSeq-count v0.6.1p1 using the parameters: --format=bam --minaqual=2 --stranded=no -i gene_id. Genes with at least 3 tag counts per 1x10^6 reads in at least two separate samples were retained for further analysis. Normalization, dispersion estimation and differential expression testing were all done using functions contained in the edgeR_3.10.5 Bioconductor package. The segment, time point, and sample batch (rep#) were included in the design model matrix (~0+seg:dayt+batch:rep). For each time point, the abundance of each gene in regenerating anterior or posterior segments was compared to the abundance in age-matched, sibling, non-bisected control larvae and significance was assessed using the quasi-liklihood F test (glmQLFit).
Genome_build: Pmin_1.0
Supplementary_files_format_and_content: Tab-delimited text files include sequence tag counts for each sample to the assembled genes as well as the output of the edgeR differential expression analysis for expressed genes, specifically the log2-transformed fold-change in gene expression at each timepoint in regeneration compared with control segments.
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| Submission date |
Mar 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Cary |
| Organization name |
Carnegie Mellon University
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| Department |
Biological Sciences
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| Street address |
4400 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
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| Platform ID |
GPL23240 |
| Series (1) |
| GSE97230 |
Regeneration time course of a larval sea star, Patiria miniata |
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| Relations |
| BioSample |
SAMN06661554 |
| SRA |
SRX2692945 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2559968_ANT-6d-r2_htseq-count.txt.gz |
277.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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