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Sample GSM6268548 Query DataSets for GSM6268548
Status Public on Sep 13, 2022
Title Stage 9 rep A2
Sample type SRA
 
Source name Whole embryos
Organism Xenopus laevis
Characteristics tissue: Whole embryos
strain: Xla.NXR-WTNXR
genotype: WT
developmental stage: NF9
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description s9_a_2
Phelps23_exon_counts.csv
Phelps23_intron_counts.csv
Phelps23_exon_tpm.csv
Phelps23_intron_rpkm.csv
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
 
Submission date Jun 27, 2022
Last update date Sep 21, 2023
Contact name Miler T Lee
E-mail(s) miler@pitt.edu
Organization name University of Pittsburgh
Department Biological Sciences
Street address 4249 Fifth Ave
City Pittsburgh
State/province PA
ZIP/Postal code 15260
Country USA
 
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
Relations
BioSample SAMN29378310
SRA SRX15911810

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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