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Sample GSM7910913

Query DataSets for GSM7910913

Status

Public on Jun 17, 2024

Title

Alim Diapause Day 6 ATACseq Rep2

Sample type

SRA

Source name

Dissected Embryo (Whole Body)

Organism

Austrofundulus limnaeus

Characteristics

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

Treatment protocol

All collected embryos were wildtype and untrated for the duration of their development.

Growth 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. 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. 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. we next monitored the onset of heartbeat, and stage diapause embryos at 6 days (Dia 6d) and 1 month of diapause (Dia 1m) as exhibiting a continuously decreasing heartbeat rate since diapause onset (<45 beat-per-minute (BPM)) as described in Hu et al. For embryos in 1 month diapause (Dia 1m), we additionally made sure that there was no heartbeat by monitoring them for 5 minutes under a stereoscope to verify that they were not prematurely exiting the diapause state. For embryos in development, embryos that had an increase in heartbeat rate 1 day after heartbeat onset (>45 BPM), but before the visual pigmentation in eyes was developed (i.e. before pharyngula stage) were designated as developing embryos (Dev). All the diapause and development stages stage are identical to our previous study, except Pre-Dia stage which is a day before the onset of heartbeat.

Extracted molecule

genomic DNA

Extraction protocol

To generate nuclei-suspension for ATAC-seq libraries, snap frozen embryo samples (~10 embryos per sample) were thawed for 1 minute and resuspended at 4oC in 200ml EZ-lysis buffer (Sigma Aldritch No. 3408). Samples were then transferred to 250 ml mini-douncers (DWK (Kimble) 885300-0000) and dounced 25 times with pestle A and B respectively. After a 2 minute incubation following douncing, samples were spun at 500g for 5 minutes to precipitate nuclei, and the EZ-lysis supernatant was removed. Nuclei were then resuspended in 250ml PBS (ThermoFisher No. AM9624) and an aliquot of 5µl of nuclei was incubated with 5ml of 0.4% trypan blue stain (ThermoFisher No. 15250061) for counting the total intact nuclei counts.
Samples of ~25,000 nuclei were then suspended in a Tn5 transposition mix (65ml of tagmentation DNA buffer (Illumina No. 20034197), 63ml of nuclease-free water, and 2.5ml of tagmentation DNA enzyme I (e.g Tn5 transposase) (Illumina No. 20034197) for 20 minutes at 37oC. Following incubation, the mix was purified using the Qiagen mini-elute kit (Qiagen No. 28206) to isolate tagmented DNA. PCR amplification and subsequent qPCR monitoring was performed as described in the original ATAC-seq protocol (~14-18 cycles of PCR). Amplified DNA from the PCR reaction was purified using the Qiagen mini-elute kit (Qiagen No. 28206), as recommended by the manufacturer. Samples were subsequently pooled and sequenced using next-generation short-read sequencing on an Illumina NEXTseq 550.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

NextSeq 550

Description

Alim_consensusPeaks_rpkm.txt
Alim_6D_2

Data processing

Fastq filles underwent read trimming using Trimgalore (v0.4.1)
Fastq files were mapped using --very-sensitve parameter on Bowtie2 (v2.2.5)
Duplicate reads were flagged and removes with Picard tools (v2.22.1) and SAMtools (v1.5)
Tn5 base shifting corrected by alignmentSieve in deepTools (version 3.2.1)
Peaks called using MACS2 (version 2.1.1.20160309)
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 ; South American Killifish: GCA_001266775.1
Supplementary files format and content: Concensus Peak files across replicates and timepoints. Columns 1-11 are Bed format entries and all following are RPKM-normalized readcounts within each sample.

Submission date

Nov 20, 2023

Last update date

Jun 17, 2024

Contact name

Anne Brunet

E-mail(s)

abrunet1@stanford.edu

Organization name

Stanford University

Department

Genetics