|
| Status |
Public on Apr 01, 2020 |
| Title |
Pt-msx1_st5_rep1_d18 |
| Sample type |
SRA |
| |
|
| Source name |
26LL
|
| Organism |
Parasteatoda tepidariorum |
| Characteristics |
isolate: Osaka
tissue: whole embryo
treatment: Pt-msx1 pRNAi
developmental stage: late stage 5
|
| Treatment protocol |
Mated adult females were injected with approximately 1-2 µl of Pt-hh, Pt-ptc, or Pt-msx1 dsRNA solution (2µg/µl) four times at 2-3 days intervals. Embryos derived from egg sacs produced by the injected females 13-24 days after the first injection of the dsRNA were used for RNA extraction. Embryos from the same females 1-3 days before the dsRNA injection were used as untreated samples.
|
| Growth protocol |
Embryos were incubated at 25˚C until they reached the appropriate stages.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Poly(A) mRNA was extracted using a QuickPrep Micro mRNA purification kit (GE Healthcare) and a Dynabeads mRNA DIRECT Kit (Ambion) from 50-120 embryos.
mRNA was fragmented using the NEBNext RNase III RNA Fragmentation Module (New England BioLabs). Sequencing libraries were constructed from fragmented RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, New England BioLabs).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
HTseq_count_per_gene_aug3.txt
HTseq_count_per_gene_aug3_RPKM.txt
DEG_msx_st5.txt
|
| Data processing |
Base call and quality scoring were performed by the Real Time Analysis software application during Illumina's sequencing runs.
Sequence reads were trimmed for adaptor and primer sequences and low-quality sequences using the CLC Genomics Workbench 7.0.4 (Qiagen) with the following parameter settings for the quality trim: trim using quality scores, limit=0.05; trim ambiguous nucleotides, maximum number of ambiguities=2; filter on length, discard reads below length=50. Trimmed reads were then aligned to Ptep_1.0 genome assembly using the BLAT algorithm version 34 in the DDBJ Read Annotation Pipeline with default settings.
The output alignments were filtered based on quality, coverage, and uniqueness using a perl script filterPSL.pl available from the AUGUSTUS 3.0.1 scripts folder. The parameter settings were as follows: minimum coverage, 60%; minimum percent identity, 90%; unique threshold, 0.96. The filtered alignments were converted into sam and psl files with a custom perl script.
Using the sam files, abundance of mRNAs against AUGUSTUS gene models (Schwager et al., 2017, BMC Biol. 15, 62) was calculated with htseq-count v. 0.5.4p5 in -s reverse, -m union settings and was normalized in Reads Per Kilobase of exon per Million reads (RPKM).
Based on the abundance of mRNAs against AUGUSTUS gene models, differentially expressed genes (DEGs) were estimated using EdgeR.
The alignments in the psl format were converted into wig files using a script aln2wig available from the AUGUSTUS 3.0.1 scripts folder, and the wig files were converted into bigWig files using the program wigToBigWig available from the ucsc directory.
Genome_build: Ptep_1.0
Supplementary_files_format_and_content: tab-delimited text file includes RPKM values for gene models and the features of the gene models.
Supplementary_files_format_and_content: tab-delimited text file includes logFC and FDR values for the identified DEGs.
Supplementary_files_format_and_content: wig files ; Scores represent normalized read coverage (per 10 million reads).
Supplementary_files_format_and_content: bigWig files ; Scores represent normalized read coverage (per 10 million reads).
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| |
|
| Submission date |
Aug 26, 2019 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Hiroki Oda |
| E-mail(s) |
hoda@brh.co.jp
|
| Organization name |
JT Biohistory Research Hall
|
| Lab |
Laboratory of Evolutionary Cell and Developmental Biology
|
| Street address |
1-1 Murasaki-cho
|
| City |
Takatsuki |
| State/province |
Osaka |
| ZIP/Postal code |
569-1125 |
| Country |
Japan |
| |
|
| Platform ID |
GPL24841 |
| Series (1) |
| GSE136357 |
Genome-wide Identification of genes that transcriptionally respond to altered states of Hedgehog signaling in embryos of the spider Parasteatoda tepidariorum |
|
| Relations |
| BioSample |
SAMN12637638 |
| SRA |
SRX6772921 |
