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| Status |
Public on Jul 20, 2021 |
| Title |
BRP241R |
| Sample type |
SRA |
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| Source name |
mature
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Trophectoderm
developmental stage: Blastocyst
day of development: Day 5
maternal age: at least 40 years old
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| Growth protocol |
Intracytoplasmic sperm injection (ICSI) was used to fertilise human oocytes. The embryos were cultured to the blastocyst stage, in continuous single culture media with 5% Human Serum Albumin (HSA) (IrvineScientific, CA, USA).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
A small hole was opened in the zona pellucida opposite the inner cell mass, using a non-contact laser. Six to eight trophectoderm cells were biopsied, inserted into lysis buffer with RNase Inhibitor (Clontech, USA) and frozen at -80°C.
RNA libraries were prepared for sequencing using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) and Nextera XT DNA library preparation kit (Illumina), according to the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Trim galore was used to trim paired-end reads and remove adapters and poor quality sequence.
HISAT2 aligner (version 2.0.4) was used to map read mates to the Human reference genome (hg38), using the following parameters -p 8 -k 1 --dta --rna-strandness FR --no-mixed --no-discordant.
Incorrectly paired and unmapped reads were removed. The remaining reads converted to bam format with the view function of samtools (version 1.3).
Picard MarkDuplicates (version 2.1.1) was emloyed to tag potential PCR duplicates.
Using the UCSC hg38 annotation of the human genome as a guide, StringTie was used to identify potential novel transcripts.
Rsubread's featureCounts function and Python script prepDE.py (https://ccb.jhu.edu/software/stringtie/dl/prepDE.py) were used to count assigned reads to genomic features. Only correctly paired-end, uniquely mapped, non-duplicated reads were considered.
The libraries were normalised for library sizes using the edgeR function calcNormFactors, that is based on the trimmed mean M value (TMM). The threshold of copies-per-million (cpm) was set at 1 for at least half of the libraries to be included in the analysis. The differential gene expression between blastocysts deriving from young and advanced maternal age women, was statistically tested by the edgeR Exact test (FDR 0.05).
Genome_build: hg38
Supplementary_files_format_and_content: Trophectoderm_GEO_refSeq_submission.xlsx: Excel file includes raw counts.
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| Submission date |
Jul 01, 2019 |
| Last update date |
Jul 20, 2021 |
| Contact name |
Panagiotis Ntostis |
| Organization name |
University of Leeds
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| Department |
Medicine and Health
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| Lab |
LICAMM
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| Street address |
Clarendon Way
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| City |
Leeds |
| ZIP/Postal code |
LS2 9JT |
| Country |
United Kingdom |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE133592 |
The impact of maternal age on trophectoderm gene expression profiles in human. |
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| Relations |
| BioSample |
SAMN12172582 |
| SRA |
SRX6385325 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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