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| Status |
Public on May 14, 2019 |
| Title |
Wnt-LOF_st9p_polyA-RNA_rep1 |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Xenopus tropicalis |
| Characteristics |
strain: wild type (out-bred Nigerian)
developmental stage: 9+ (late blastula)
treatment: injection of beta-catenin MO (canonical Wnt inhbition)
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| Treatment protocol |
Injections were carried out into the zygote's animal pole: β-catenin MO (5 ng); 4x zT-box MOs (5 ng, zVegT MO, 5 ng Eomes MO, 2.5 ng Xbra.1 MO, 2.5 ng Xbra.2 MO, 2.5 ng Xbra3.1 MO and 2.5 Xbra3.2 MO); and standard control MO (5 or 20 ng according to dose used for the loss-of-function experiment). Embryos were treated with antagonists of Nodal and/or BMP signaling at the following concentrations in 5% MMR: 100 µM SB431542 and/or 10 µM LDN19318 from the 8-cell stage onwards. Control embryos were treated accordingly with DMSO, the solvent of these antagonists.
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| Growth protocol |
Embryos were grown to the indicated developmental stage in 5% MMR at 25ºC.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA extraction: 10-15 dejellied X. tropicalis embryos were homogenized in 800 µl TRIzol reagent by vortexing. For phase separation, the homogenate supplemented with 1/5 volume of chloroform was transferred to a 1.5-ml phase-lock gel heavy microcentrifuge tube, shaken vigorously for 15 sec, left on the bench for 2 min and centrifuged (16,000 g) for 5 min at 4ºC. The upper phase was mixed with 1 volume of absolute ethanol and centrifuged (12,000 g) through the column IIC of Zymo RNA Clean and Concentrator-25 (Zymo Research) for 30 sec at room temperature. Next, the manufacturer’s instructions were followed for the recovery of total RNA (>17 nt) with minor modifications. First, the flow-through of the first spin was re-applied to the column. Second, the RNA was treated in-column with Turbo DNase (2 U/µl, ThermoFisher Scientific). Third, the RNA was eluted twice with 25 µl molecular-grade water. The concentration was determined on the Nanodrop 1000 except for RNA samples subjected to ribosomal RNA depletion in which case more accurate fluorometry was used.
Metabolic labeling of nascent transcripts: 75 ng 4-thiouridine-5'-triphosphate (4sU) was injected into the zygote’s animal pole. Total RNA was extracted as described above from 30 embryos collected at the mid-blastula transition (stage 8+) and early gastrula stage (stage 10+). Ribosomal RNA (rRNA) was removed from ~10 µg total RNA with Ribo-Zero rRNA (human/mouse/rat) magnetic beads (Illumina) following the manufacturer’s instructions. rRNA-depleted total RNA was purified with the RNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer’s instructions. The yield was determined on a fluorometer using high-sensitivity reagents for RNA (250 pg/µl to 100 ng/µl). The purified RNA was transferred to thin-walled PCR tubes and fragmented with 40 µl 1x NEBNext RNA fragmentation buffer (NEB) in a thermocycler for 4 min at 95ºC. The fragmentation was stopped on ice with NEBNext RNA fragmentation stop solution (NEB) before purifying the fragmented RNA with RNA Clean & Concentrator-5. The RNA was eluted twice with 15 µl molecular-grade water. Approximately 10 ng of RNA was set apart to make an input library. The remaining RNA was biotinylated in the dark with EZ-Link HPDP-Biotin for 3 h at room temperature. The RNA was purified with RNA Clean & Concentrator-5 and eluted twice with 20 µl molecular-grade water. Biotinylated RNA was separated from non-labelled RNA with M270 streptavidin magnetic beads following the manufacturer’s instructions. The biotinylated RNA was stripped off the beads twice with 100 µl 100 mM 2-mercaptoethanol (pre-heated to 80ºC) and purified with RNA Clean & Concentrator-5.
RNA-Seq: Indexed paired-end libraries were made from ~1 µg total RNA by following the low sample protocol of the TruSeq RNA sample preparation guide v2 with a few modifications. First, cDNA after second strand synthesis was quantified using a fluorometer and high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl). Second, the number of PCR cycles was adjusted to the detected yield of cDNA to avoid products of over-amplification such as chimera fragments: 7 (~20 ng), 8 (~10 ng), 9 (~5 ng), 12 (~1 ng).
4sU-Seq: Input and biotinylated RNA were converted to indexed paired-end libraries using the ScriptSeq v2 RNA-Seq preparation kit (Epicentre). We used the manufacturer’s instructions (protocol rev. C, 8/2014) starting with appendix 4.1.A (annealing cDNA synthesis primer) and 4.1.B (cDNA synthesis) followed by main protocol section 3.C (synthesis of 3’-tagged DNA). The DNA libraries were purified using 1.8x Agencourt AMPure XP beads and amplified with 11 (input) and 15 (streptavidin pull-down) PCR cycles according to section 3.D to 3.F. The DNA from the PCR reaction was purified using 1x AMPure XP beads and eluted in 10 µl molecular-grade water.
Illumina libraries were sequenced paired-end on the HiSeq 2500 (4sU-Seq) and HiSeq 4000 (RNA-Seq).
RNA-Seq: Indexed paired-end libraries were made from ~1 µg total RNA by following the low sample protocol of the TruSeq RNA sample preparation guide v2 with a few modifications. First, cDNA after second strand synthesis was quantified using a fluorometer
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Wnt loss-of-function, stage 9+ (late blastula), experiment 4
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| Data processing |
Bases were called using Casava version 1.8.2
RNA-Seq: Paired-end reads were aligned to the X. tropicalis genome assembly v7.1 using STAR v2.5.3a with default settings and a revised version of gene models v7.2 to improve mapping accuracy across splice junctions. The alignments were sorted by read name using the sort function of samtools v1.3.1. Exon and intron counts (-t 'exon;intron') were extracted from unstranded (-s 0) alignment files using VERSE v0.1.5 in featureCounts (default) mode (-z 0). Intron coordinates were adjusted to exclude any overlap with exon annotation. Differential expression analysis was performed with both raw exon and intron counts excluding those belonging to ribosomal and mitochondrial RNA using the R package DESeq2 v1.14.1. In an effort to find genes with consistent fold changes over time, p-values were generated according to a likelihood ratio test reflecting the probability of rejecting the reduced (~ developmental stage) over the full (~ developmental stage + condition) model. Resulting p-values were adjusted to obtain false discovery rates (FDR) according to the Benjamini-Hochburg procedure with thresholds on Cook’s distances and independent filtering being switched off. Equally, dataset without time series were subjected to likelihood ratio tests with reduced (~1) and full (~ condition) models for statistical analysis.
4sU-Seq: Paired-end reads were aligned to a revised version of X. tropicalis gene models v7.2 and known off-genome EST assemblies including ribosomal and mitochondrial RNA by running Bowtie2 v2.1.0 with the following constraints: -k 200 (up to 200 alignments per fragment) -X 800 (maximum fragment length of 800 bp) -rdg 6,5 (penalty for read gaps of length N, 6+N*5) -rfg 6,5 (penalty for reference gaps of length N, 6+N*5) -score-main L,-.6,-.4 (minimal alignment score as a linear function of the read length x, f(x) = -0.6 - 0.4*x) -no-discordant (no paired-end read alignments breaching maximum fragment length X) -no-mixed (only concordant alignment of paired-end reads). Only read pairs that uniquely align to one gene were counted. Differential expression analysis was performed with raw read counts including those belonging to ribosomal and mitochondrial RNA using the R package DESeq2 v1.14.1. In an effort to find 4sU-enriched transcript levels, their DESeq2 normalised read counts genes were divided by those of the 4sU input for both developmental stages.
Genome_build: X. tropicalis v7.1
Supplementary_files_format_and_content: Raw read count tables (gzip compressed text files)
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| Submission date |
Nov 14, 2018 |
| Last update date |
May 15, 2019 |
| Contact name |
George E. Gentsch |
| E-mail(s) |
george.gentsch@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| Department |
Developmental Biology Laboratory
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| Lab |
James C. Smith
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| Street address |
1 Midland Road
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| City |
London |
| State/province |
Greater London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platform ID |
GPL23182 |
| Series (1) |
| GSE122551 |
The Spatiotemporal Control of Zygotic Genome Activation |
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| Relations |
| BioSample |
SAMN10424677 |
| SRA |
SRX5008753 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3473724_WntLOF_st9p_frogG_GG135.exon.txt.gz |
104.6 Kb |
(ftp)(http) |
TXT |
| GSM3473724_WntLOF_st9p_frogG_GG135.intron.txt.gz |
77.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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