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Sample GSM8181468

Query DataSets for GSM8181468

Status

Public on Aug 06, 2024

Title

sp_2dpf_3_scRNAseq

Sample type

SRA

Source name

dissociated 2dpf gastrula embryo

Organism

Strongylocentrotus purpuratus

Characteristics

tissue: dissociated 2dpf gastrula embryo
developmental stage: 2dpf gastrula
time: 2 dpf
cell type: 2dpf gastrula cells

Growth protocol

AdultStrongylocentrotus purpuratus were kept in circulating seawater aquaria at Stazione Zoologica Anton Dohrn in Naples. Spawning was induced by vigorous shaking of adult animals. Embryos were cultured at 15 °C in filtered Mediterranean Sea water diluted 9:1 with deionized water.

Extracted molecule

polyA RNA

Extraction protocol

For scRNAseq embryos were collected using a 40 μm Nitex mesh filter and spun down at 500 g for 5 min. Sea water was removed and embryos were resuspended in Ca2+ Mg2+-free artificial sea water, then spun down at 500 g for 5 min and resuspended in dissociation buffer (1 M glycine and 0.02 M EDTA in Ca2+ Mg2+-free artificial sea water). Embryos were incubated for 10 min on ice and dissociated gently via pipette aspiration. Dissociated cells were spun down at 700 g for 5 min and washed several times with Ca2+ Mg2+-free artificial sea water.
scRNA-seq: Single cell cDNA libraries were prepared using the Chromium Single Cell 3’ Reagent Kit, Chemistry v3 and v3.1.
scRNA-seq: Libraries were indexed, multiplexed and sequenced on Illumina NextSeq 500 to generate paired end reads.
For ATACseq Embryos were collected using a 40 μm Nitex mesh filter and spun down at 500 g for 5 min. For gut tissue sea water was removed and embryos were resuspended in Ca2+ Mg2+-free artificial sea water, then spun down at 500 g for 5 min and resuspended in dissociation buffer (1 M glycine and 0.02 M EDTA in Ca2+ Mg2+-free artificial sea water); embryos were incubated for 10 min on ice and then continuously pipetted up and down with P1000 micropipette to mechanically separate the gut tissue. Separated guts were collected in 100 μl of artificial sea water on ice. The guts or whole embryos were washed in 4 °C artificial sea water, the lysed in 50 μl of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% IGEPAL CA-630 lysis buffer. Nuclei were centrifuged at 500g, supernatant discarded. 75000 nuclei were tagmented in 2x tagmentation buffer (20 mM Tris-HCl, 10 mM MgCl2, 20% (vol/vol) dimethylformamide), 23.75 μl of nuclease free water and 1.25 μl of Tn5 enzyme from Dr Jose Luis Gómez-Skarmeta.
ATAC-seq: The tagmented DNA was purified using MinElute Kit (Qiagen) and eluted in 10 μl of elution buffer, and amplified (10 μl of eluted tagmented DNA, 10 μl of nuclease free water, 2.5 μl 10 μM Nextera Primer 1, 2.5 μl 10 μM Nextera Primer 2.X with following program: 72°C for 5 min, 98°C for 30 sec, then 15 cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min, followed by a hold step at 4°C). The resulting library was purified using MinElute Kit (Qiagen) and eluted in 20 μl of elution buffer.
ATAC-seq: Indexed libraries were multiplexed and sequenced on Illumina HiSeq 4000 at BGI Tech (HongKong) to generate paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

sp_2dpf_integrated_umap.rds

Data processing

scRNA-seq:
scRNAseq FASTQ reads were aligned to S. purpuratus genome version 3.1 using Cell Ranger 3.0.2 (10x Genomics).
Cell Ranger output files for all the replicates were used in Seurat v3.0.2 R package.
Genes transcribed in less than three cells and cells with less than 200 transcribed genes were excluded.
Datasets were normalized and variable genes were found using vst method with maximum 2000 variable features.
Data was integration was performded using anchors between the six different objects.
Data was scaled and principal component (PCA) analysis was performed. Sharing Nearest Neighbor (SNN) was computed with 20 dimensions (resolution 1.0). UMAP was used to perform clustering dimensionality reduction.
Positive cluster markers were found using the genes that are detected in at least 0.01 fraction of min.pct cells.
ATAC-seq:
ATACseq FASTQ reads were trimmed using Trimmomatic 0.38 with ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:6 CROP:40 SLIDINGWINDOW:3:25 MINLEN:25 settings.
Trimmed reads were mapped to S. purpuratus genome version 3.1 using bowtie2 2.3.4.1.
Resulting bam files were filtered to have fragments of less than 130, with quality over 30, mates were fixedd and duplicates removed using samtools 1.7.1. Then bam files were converted into bed using bedtools 2.27.1.
MACS2 2.1.2 was used for peak-calling with --extsize 100, --shift 50, --nomodel settings with genome size of 815936258.
Bedtools 2.27.1 intersect and merge were used to keep regions in both replicates per sample and merge peaks from gut and whole embryo samples.
Assembly: S. purpuratus genome 3.1
Supplementary files format and content: R object file, tab separated values

Submission date

Apr 01, 2024

Last update date

Aug 06, 2024

Contact name

Maria Ina Arnone

E-mail(s)

miarnone@szn.it

Organization name

Stazione Zoologica Anton Dohrn

Department

Biology and Evolution of Marine Organisms