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| Status |
Public on Sep 13, 2022 |
| Title |
Stage 5 (COX2, COX3 deplete) rep 1 |
| Sample type |
SRA |
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| Source name |
Whole embryos
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| Organism |
Xenopus laevis |
| Characteristics |
tissue: Whole embryos
strain: Xla.NXR-WTNXR
genotype: WT
developmental stage: NF5
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| Treatment protocol |
Dejellied embryos were incubated in MR/3 (33 mM NaCl, 0.6 mM KCl, 0.67 mM CaCl2, 0.33 mM MgCl2, 1.67 mM HEPES, pH 7.8) at 23C and collected at their respective NF stages based on morphology. All stage 9 embryos were collected halfway through the stage, at 8 hours post fertilization. Triptolide samples were bathed in 20 uM triptolide in DMSO at stage 1 and cycloheximide samples were bathed in 500 ug/mL cycloheximide in DMSO at stage 8; both were collected when batch-matched, untreated embryos reached stage 9. Equivalent volumes of DMSO were used to treat control samples. Morpholino treated embryos were injected at stage 1 with pou5f3.2, pou5f3.3, sox3, and/or GFP control morpholino.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Two embryos per sample were snap frozen and homogenized in 500ul of TRIzol Reagent (Invitrogen #15596026) followed by 100ul of chloroform. Tubes were spun at 18,000 x g at 4C for 15 minutes, the aqueous phase was transferred to a fresh tube with 340 ul of isopropanol and 1 ul of GlycoBlue (Invitrogen #AM9515), then precipitated at -20C overnight. Precipitated RNA was washed with cold 75% ethanol and resuspended in 50ul of nuclease-free water. Concentration was determined by NanoDrop.
rRNA depletion was performed as per Phelps et al NAR 2021: 1ul of antisense nuclear rRNA oligos and 1ul of antisense mitochondrial rRNA oligos (final concentration 0.1 uM per oligo) were combined with 1ug of total RNA in a 10ul buffered reaction volume (100mM Tris-HCl pH 7.4, 200mM NaCl, 10mM DTT), heated at 95C for 2 minutes and cooled to 22C at a rate of 0.1C/s in a thermocycler. Next, 10U of thermostable RNaseH (NEB #M0523S) and 2uL of provided 10X RNaseH buffer were added and volume brought to 20uL with nuclease-free water. The reaction was incubated at 65C for 5 or 30 minutes, then 5U of TURBO DNase (Invitrogen #AM2238) and 5uL of provided 10x buffer was added, volume brought to 50uL with nuclease-free water and incubated at 37C for 30 minutes. The reaction was purified and size selected to >200 nts using Zymo RNA Clean and Concentrator-5 (Zymo #R1013) according to manufacturer’s protocol, eluting in 10uL of nuclease-free water. The WT Stage 5 sample was also depleted of mitochondrial COX2 and COX3 mRNA as part of Phelps et al NAR 2021. Strand-specific RNA-seq libraries were constructed using NEB Ultra II RNA-seq library kit (NEB #E7765) according to manufacturer’s protocol with fragmentation in first-strand buffer at 94C for 15 minutes. Following first and second strand synthesis, DNA was purified with 1.8X AmpureXP beads (Beckman #A63880), end repaired, then ligated to sequencing adaptors diluted 1:5. Ligated DNA was purified with 0.9X AmpureXP beads and PCR amplified for 8 cycles, then purified again with 0.9X AmpureXP beads. Libraries were verified by Qubit dsDNA high sensitivity (Invitrogen #Q32851) and Fragment Analyzer prior to multiplexed sequencing at the Health Sciences Sequencing Core at Children’s Hospital of Pittsburgh.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
s5_1
Phelps23_exon_counts.csv
Phelps23_intron_counts.csv
Phelps23_exon_tpm.csv
Phelps23_intron_rpkm.csv
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| Data processing |
Reads were mapped to the X. laevis v9.2 genome using HISAT2 v2.2.1 (--no-discordant)
Mapped reads were assigned to gene exons (Xenbase v9.2 models) using featureCounts v2.0.1 in reversely-stranded paired-end mode with default parameters, or introns with --minOverlap 10 on a custom non-repeat non-exonic annotation based on Xenbase models
Assembly: X. laevis v9.2
Supplementary files format and content: CSV exon and intron raw and normalized read counts
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| Submission date |
Jun 27, 2022 |
| Last update date |
Sep 21, 2023 |
| Contact name |
Miler T Lee |
| E-mail(s) |
miler@pitt.edu
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| Organization name |
University of Pittsburgh
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| Department |
Biological Sciences
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| Street address |
4249 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15260 |
| Country |
USA |
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| Platform ID |
GPL21248 |
| Series (2) |
| GSE207026 |
Hybridization led to a rewired pluripotency network in Xenopus laevis [RNA-seq] |
| GSE207027 |
Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis |
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| Relations |
| BioSample |
SAMN29378316 |
| SRA |
SRX15911804 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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