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Sample GSM8024811

Query DataSets for GSM8024811

Status

Public on Jan 06, 2025

Title

JC_1_113 (21PG37-2)

Sample type

SRA

Source name

embryo

Organism

Cavia porcellus

Characteristics

tissue: embryo
developmental stage: Early blastocyst
time: embryo in vitro day E5.25

Treatment protocol

Embryos at each stage were retrieved from at least 3 separate Hartley guinea pig females (~500 grams, 2-3 months of age) between days 3.5 and 5.75 after natural mating.

Growth protocol

Hartley guinea pigs (Charles River Labs, Laval, Québec) were housed in the Animal Care Facility at the CRCHUM, maintained at 21 °C with 30-70 % humidity under a 12 h light cycle, and fed with Teklad Global Guinea Pig Diet 2040 pellets, water, and hay ad libitum.

Extracted molecule

polyA RNA

Extraction protocol

Briefly, embryos were flushed from the oviduct (E3.5) or uterine horns (E4-E5.75) using 5-10 ml of warm M2 medium (MR-015-D, Sigma Aldrich) supplemented with 1 % Penicillin-Streptomycin (15130122, Life Technologies), washed and briefly kept in M2 drops covered with Ovoil TM (10029, Vitrolife) at the incubator at 5 % O2 5 % CO2 at 37.5 °C prior to being fixed in PFA 4 % for 15 min or single cell picking. E6 embryos were collected at E5.75 and incubated in similar conditions for 6 h to reach the desirable embryonic day, as implantation occurs around E6, and obtaining embryos by flushing the uterus was challenging.
Embryos collected from E3-E5.75 were obtained through the flushing female guinea pigs and placed in M2 drops under mineral oil until further processing. Zona pellucida was removed using tyrodes, and subsequent single-cell dissociation was achieved through enzymatic digestion with TrypLE Express (12604-013, Gibco, Life Technologies) followed by isolation using fine glass capillaries. Libraries were prepared using the Smartseq2 protocol, following established procedures 14–16. The quantity and quality of the cDNA libraries were assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies). Approximately 1 ng of cDNA per cell was transformed into a single-cell library using the Nextera XT DNA Library prep kit (Illumina FC-131-1096) and following the manual instructions with slight modifications. In brief, 1 ng of cDNA (in a maximum of 5 µl of sample volume) underwent tagmentation with 2 µl of TD and 1 µl of ATM at 55 °C for 5 min. Upon completion of the program, the reaction was promptly halted by adding 1 µl of 2 % SDS and incubating for 5 min at RT. Amplification was carried out using 3 µl of NMP per sample and adding 1 µl of each dual-indexed (i7 and i5; Illumina) primer. Individual Nextera XT libraries were pooled and then purified using magnetic beads. Indexed sequence libraries were combined for multiplexing (384 samples per lane). The sequencing was performed using the NovoSeq6000 paired-end.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Gpig.scRNAseq.counts.all.tsv.gz

Data processing

For the guinea pig single-cell RNA-Seq data, adapters and low-quality reads were removed using Trimgalore with default parameters. Subsequently, high-quality reads were aligned to the guinea pig Ensembl reference genome (v.Cavpor.3.0.105, Cavia porcellus, obtained from the Ensembl website)using the HISAT2 aligner (v 2.2.0) with default settings. Only uniquely mapped reads were retained for gene expression quantification. Raw read counts were tentatively quantified using featureCounts (v1.6.3) based on Ensembl reference annotation.
Considering the incomplete guinea pig reference annotation, we decided to update the guinea pig gene annotation using the following steps: 1. Ensembl transcripts belonging to “lincRNA”, “processed_pseudogene”, “protein_coding”, and “pseudogenes” were selected as the guided annotation. The mapping bam files were used as input for Stringtie (v1.3.3b) for each individual sample. After the initial assembly, the transcripts were merged using 'stringtie --merge' and compared with the guided annotation using 'gffcompare -r'. Transcripts that overlapped with multiple reference genes were excluded, as well as transcripts with a length less than 200 bp and 'class codes' identified by gffcompare equal to ‘e’, ‘s’, ‘x’ were removed in downstream analysis. Transcripts with class codes 'j', 'k', '=', 'm', 'n', 'o', 'p', 'y' were assigned to corresponding reference genes. Novel transcripts with no clear reference annotation were blasted against human and mouse transcripts of protein coding and lncRNA with parameters '-max_target_seqs 1 -outfmt 6 -num_threads 25 -evalue 1e-20 -strand plus -max_hsps 1' to find the most similar human or mouse reference genes. If there were no orthologous genes of matched reference genes in the guinea pig Ensembl annotation, they were added. Other novel transcripts with known orthologous genes in the guinea pig Ensembl annotation were reassigned to known annotated genes based on whether the novel transcripts fell within the nearby loci (± 3 kb) of the current annotation.
Assembly: Cavpor.3.0.105
Supplementary files format and content: tab-delimited text file include raw count for each Sample

Submission date

Jan 19, 2024

Last update date

Jan 06, 2025

Contact name

Cheng Zhao

E-mail(s)

zhaocheng3326@gmail.com

Organization name

Karolinska institute

Department

Clinical Science