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| Status |
Public on Jun 04, 2024 |
| Title |
pool of embryo, low concentration of P4, rep1 |
| Sample type |
SRA |
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| Source name |
pool of embryo
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| Organism |
Bos taurus |
| Characteristics |
tissue: pool of embryo
treatment: low concentration of P4
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| Extracted molecule |
total RNA |
| Extraction protocol |
Embryo total RNA was extracted from each biological replicate using the PicoPure RNA isolation kit (Thermo Fisher Scientific) with DNAse treatment (Qiagen) as per the manufacturer’s protocol. RNA was eluted in 15 µL of nuclease-free water and kept at -80°C for subsequent library preparation. RNA concentration and integrity were evaluated using an Agilent 4200 TapeStation (Agilent Technologies) with the High Sensitivity RNA ScreenTape Assay (Agilent Technologies). RNA Integrity Number (RIN) ranged from 5.0 to 8.3 RIN with an average of 6.2 ± 1.4 RIN and RNA concentration ranged from 308 to 556 pg/µL with an average of 414 ± 113.6 pg/µL.
Library preparation was performed for each biological replicate. Enrichment of mRNA was conducted using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, New England Biolabs, USA) and libraries were prepared using the NEBNext Ultra II RNA Library kit for Illumina (E7770, E7775, New England Biolabs, USA). The library preparation protocol was followed as recommended by the manufacturer’s guidelines for 10 ng – 1μg of DNA-free total RNA, with 15 cycles for the PCR enrichment of adaptor-ligated DNA and Illumina index primers (Illumina). Purified libraries were eluted in 23 μL of (0.1X) TE buffer and stored at -20°C for future analysis. The quality of the 6 libraries were verified using Agilent bioanalyzer (4200 TapeStation, Agilent Technologies) with the High Sensitivity D1000 ScreenTape Assay (Agilent Technologies) before libraries were pooled in equimolar proportion and sequenced with NovaSeq 6000 using paired-end 100bp protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
LP1
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| Data processing |
Preprocessing of the raw sequencing data was conducted using Trimmomatic. First, the Illumina adapters were removed. The quality threshold of 30 was set and base calls with a score below the threshold were removed from the end or/and start of a read. Trimming was performed with the minimal length set at 32 nt and reads were retained for further processing. Pseudoalignment was carried out using Kallisto and quantification of all transcripts described in release 105 of ENSEMBL cDNA gene annotation was estimated.
Pairwise comparisons in EdgeR was used to identify differential expression of genes between treatment groups. Normalized data were sorted for significance and filtered as per the Transcript Support Level (TSL) used by the Ensembl gene annotation system. Briefly, mRNA and EST alignments were compared to the GENCODE transcripts and transcripts were scored according to how well the alignment matches over its full length. This approach was used to highlight the well supported and poorly supported transcript models. Selected differentially expressed genes were assigned to the evaluated annotation tsl1 and all splice junctions of the transcript were supported by at least one non-suspect mRNA. Due to multiple comparisons, adjusted P-values of differential expression between treatment groups were calculated using the Benjamini-Hochberg correction which controlled for a False Discovery Rate (FDR) of 5%
Assembly: release 105 of ENSEMBL cDNA gene annotation Bos taurus ARS-UCD1.2
Supplementary files format and content: comma-delimited csv file containing ensemble gene id, gene symbol, and raw counts for each sample
Supplementary files format and content: comma-delimited csv file containing ensemble gene id, gene symbol, and normalized count (tpm) for each sample
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| Submission date |
Dec 05, 2023 |
| Last update date |
Jun 04, 2024 |
| Contact name |
Marc-André Sirard |
| E-mail(s) |
Marc-Andre.Sirard@fsaa.ulaval.ca
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| Organization name |
Université Laval
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| Department |
Sciences Animales
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| Street address |
Offfice 2732, 2440 Hochelaga Blvd.
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| City |
Québec City |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
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| Platform ID |
GPL26012 |
| Series (1) |
| GSE249344 |
Embryo response to different progesterone concentrations during superovulation of Holstein heifers |
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| Relations |
| BioSample |
SAMN38658937 |
| SRA |
SRX22756893 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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