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Sample GSM7899869

Query DataSets for GSM7899869

Status

Public on Jun 17, 2024

Title

Nfur FOXO3a-KO 6-Day Diapause RNAseq Rep2

Sample type

SRA

Source name

Dissected Embryo (Whole Body)

Organism

Nothobranchius furzeri

Characteristics

tissue: Dissected Embryo (Whole body)
Stage: Diapause
timing: 6-Day Diapause

Treatment protocol

Synchronized killifish embryos for African turquoise and South American killifish were collected within a tight (~5 hour) breeding window. Most collected embryos were at the 1-2 cell stage upon collection. We monitored embryos every day post-collection to observe the visual markers of diapause and development as previously described (12). Briefly, we used Kupffer’s vesicle (KV), which is a transient embryonic organ present from early to middle somitogenesis as a marker to stage embryos that are about to reach diapause. KV-positive embryos reach the end of somitogenesis in 1-2 days and the loss of KV roughly coincides with the onset of heartbeat in killifish, followed by either diapause or continue development (12, 17). We counted the number of somites in KV-positive embryos and designated KV-positive embryos at 15-22 somites as our “pre-diapause (Pre-Dia) stage”. Embryo morphology for all the killifish species was similar at this stage. This mid-somitogenesis time point also coincides with the vertebrate phylotypic period (the period of the most conserved gene expression pattern during vertebrate development) with available gene expression and chromatin accessibility data from multiple other fish species

Growth protocol

Animals were housed in automated circulating water system with pH maintained at 6-7.5 and conductivity maintained between 3500 and 4500μS/cm with a 10% system water exchange every day by reverse osmosis treated water. Adult fish were manually fed Otohime fish diet (Reed Mariculture, Otohime C1 [Ep1 for the South American killifish]) twice a day during weekdays and once a day during weekends.

Extracted molecule

total RNA

Extraction protocol

For each stage in each species, roughly 8-30 embryos were carefully dissected in ice-cold PBS using biological-grade tweezers (Electron Microscopy Sciences, 72700-D) to carefully remove the chorion, the enveloping layer, and the yolk without damaging the embryo body. Freshly dissected embryos were then quickly rinsed with ice-cold PBS, and all the PBS was carefully removed. Embryo bodies were then snap-frozen in liquid nitrogen and stored at -80°C. We used 8-10 snap-frozen embryos for RNA-seq. Snap frozen embryos at -80°C were thawed on ice for 1 minute and washed with 200µl ice-cold PBS. The embryos were then dissociated and homogenized with ~25 Zirconia/Silicon 0.5mm glass beads (RPI, Research Products International Corp, 9834) using FastPrep® -24 homogenizer (MB Biomedicals, 116004500) for 20 seconds, followed by centrifugation (17000g for 3 minutes). After centrifugation, 10.5µl of the supernatant was used as input to the SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara, 634890) for the cDNA synthesis followed by amplification with 12 cDNA amplification cycles. Amplified cDNA was validated with Agilent 2100 Bioanalyzer using Agilent’s High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).
The DNA libraries were then generated using the Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096). Library quality and concentration were assessed by the Agilent 2100 Bioanalyzer and Agilent’s High Sensitivity DNA Kit (Agilent Technologies, Cat. No. 5067-4626), followed by high throughput sequencing on Illumina HiSeq platform with 2 x 150bp paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Nfur_FOXO3a_6D_2
Nfur_KO_Readcounts.csv

Data processing

read trimming with Trim Galore (version 0.4.5)
Genome Alignment using STAR (version 2.7.1a) No reference genome is available for the red-striped killifish, so the reads from red-striped killifish RNA-seq libraries were aligned to the genome of its close relative, lyretail killifish.
Keep only uniquely mapped reads using samtools (version 1.5) samtools view -q255
Readcounts obtained using feature counts function from Subread package (version 2.0.1)
Normalized expression using DESeq2 (version1.30.1)
Assembly: Genomes used: African Turqouise Killifish: GCA_001465895.2 ; Red-Striped & Lyretail Killifish: GCA_006937985.1 MPIBA_Aaus_1.0 scaffolds: 12,236 contigs: 30,782 N50: 119,465 L50: 1,783
Supplementary files format and content: Read counts files for each speacies from featurecount of Subread package

Submission date

Nov 14, 2023

Last update date

Jun 17, 2024

Contact name

Anne Brunet

E-mail(s)

[email protected]

Organization name

Stanford University

Department

Genetics