|
| Status |
Public on Dec 01, 2016 |
| Title |
PmTbr MASO rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Pooled Embryos
|
| Organism |
Patiria miniata |
| Characteristics |
developmental stage: hatched blastula
age: 36 hpf
morpholino: Pm-Tbr
|
| Treatment protocol |
Patiria miniata zygotes were injected with 600 µM Tbr MASO or standard control MASO (Gene Tools) as described previously. Embryos were sorted on the basis of rhodamine tracer and morphology to ensure that only healthy injected embryos were processed for RNA. Embryos that arrested in early development or that exhibited aberrant development were discarded.
|
| Growth protocol |
Embryos were cultured in artificial seawater at 15°C until hatching, approximately 30 hpf for standard control injected and typically 36 hpf for Tbr MASO injected.
|
| Extracted molecule |
total 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)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Pm-Tbr knockdown
|
| 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 2 tag counts per 1x10^6 reads in at least three 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 sample batch (rep#) and treatment were included in the design model matrix (~0+treatment+batch) and significance was assessed using the quasi-liklihood F test (glmQLFit) and controlled for errors based on multiple hypothesis testing.
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 which includes the genomic loci of each assembled gene.
|
| |
|
| Submission date |
Nov 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Cary |
| Organization name |
Carnegie Mellon University
|
| Department |
Biological Sciences
|
| Street address |
4400 Fifth Ave
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL22676 |
| Series (2) |
| GSE89863 |
RNA-seq assessment of morpholino knockdown of the sea star (Patiria miniata) Pm-Tbr transcription factor in early embryos |
| GSE89865 |
Genome-wide use of high and low affinity Tbrain transcription factor binding sites during echinoderm development |
|
| Relations |
| BioSample |
SAMN06017613 |
| SRA |
SRX2349185 |
