|
| Status |
Public on Mar 07, 2016 |
| Title |
RNA-seq sample of TBP/TBP2/TLF-AS-injected embryos_1 |
| Sample type |
SRA |
| |
|
| Source name |
10 embryos at developmental stage 10.5 (according to Nieuwkoop and Faber)
|
| Organism |
Xenopus laevis |
| Characteristics |
gene knockdown: TBP/TBP2/TLF triple knockdown (TKD)
developmental stage at injection: 1-cell stage
developmental stage at collection: stage 10.5
tissue: embryo
|
| Treatment protocol |
Embryos were dejellied in 3% cysteine, pH=8 (in 25% MMR: 22mM NaCl, 0.5mM KCl, 0.5 mM CaCl2, 0.25mM MgCl2, 1.25mM HEPES, pH 7.4). Embryos were injected with mixure of 0.8ng of TBP/TBP2/TLF AS. Embryos were cultured in 25% MMR at 18oC. At stage 10.5 (Nieuwkoop Faber) 10 embryos were collected for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol (Life Technologies) and DNA-removal was done by 'DNA-free' Kit AM1906 (Ambion). Ribosomal RNA was depleted with Ribo-Zero Magnetic Gold Kit (Epicentre).
Instructions of manufacturer were followed using KAPA Hyper Prep Kit (Illumina). Sample preparation was done using Random Hexamer primers (Roche) and Hyper Prep Kit (Illumina).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Mixture of stable antisense (AS) DNA oligonucleotides were synthesized with dimethyl-ethylene-diamine (DMED) modification (Dagle and Weeks, 2001). Sequence of TBPAS3 (7 open) Oligo 5’-T+G+C+A+T+TTCGCGCT+C+T+A+A+G-3' , TBP2-AS167 (6 open) Oligo 5’-T+C+A+T+C+GTATGGA+G+T+C+A+T-3’, TLF-AS93 (6 open) Oligo 5’-A+T+A+T+C+CAGTGCC+A+C+A+T+C-3’ (Jacobi et al., 2007; Jallow et al., 2004; Veenstra et al., 2000).
75 cyles of single-end sequencing ran on single flow of NextSeq500 (Illumina) equipment. Output data was mapped to Xenopus Genome version JGI 9.1 using STAR allowing one single mismatch. Raw data files contain 75 nucleotides long sequenses, were subjected to further analyis without additional filtering. Quality check was done by FastQ software.
|
| Data processing |
Reads were mapped to the reference X. laevis genome assembly JGI version 9.1, downloaded from Xenbase (James-Zorn et al., 2015), using STAR (Dobin et al., 2013) and allowing one mismatch.
Differential count data for each transcripts were identified using DEseq (Anders and Huber, 2010), controlling the false discovery rate at 10% using the adjusted p-value.
Genome_build: Xenopus laevis Genome version JGI 9.1. A FASTA file with transcript sequences is available on the series page of GSE76991 or at: http://www.xenbase.org/other/static/ftpDatafiles.jsp
Supplementary_files_format_and_content: Excel file contains JGI9.1 gene IDs, transcript names, base mean of counts of samples and controls: baseMeanA for knockdown, baseMeanB for controls, fold-change ratios in log10 and log2 values, and measures of statistical significance. The interpretation of the columns of data.frame is as follows. #gene_short short name of annotated gene model #id feature identifier #name whole name of annotated gene model #baseMean mean normalised counts, averaged over all samples from both conditions #baseMeanA_TKD mean normalised counts from TripleKD duplicates A #baseMeanB_ctrl mean normalised counts from water control duplicates #log2FoldChange_TKD_cntrl the logarithm (to basis 2) of the fold change #pval p value for the statistical significance of this change #padj p value adjusted for multiple testing with the Benjamini-Hochberg.
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| |
|
| Submission date |
Jan 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gert Jan C. Veenstra |
| Organization name |
Faculty of Science, Radboud Institute for Molecular Life Sciences
|
| Department |
Molecular Developmental Biology
|
| Lab |
Embryonic Gene Regulation
|
| Street address |
Geert Grooteplein 28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL21248 |
| Series (2) |
| GSE76994 |
Differential expression analysis of triple knockdown of TBP and TBP-related factors (TKD) in Xenopus laevis embryos at early developmental stage 10.5 |
| GSE76995 |
Differential expression analysis of Gcn5 knockdown and triple knockdown of TBP and TBP-related factors (TKD) in Xenopus laevis embryos at early developmental stage 10.5 |
|
| Relations |
| BioSample |
SAMN04422448 |
| SRA |
SRX1535321 |
