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Sample GSM3912668 Query DataSets for GSM3912668
Status Public on Jul 20, 2021
Title BRP241R
Sample type SRA
 
Source name mature
Organism Homo sapiens
Characteristics tissue: Trophectoderm
developmental stage: Blastocyst
day of development: Day 5
maternal age: at least 40 years old
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).
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
 
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.
 
Submission date Jul 01, 2019
Last update date Jul 20, 2021
Contact name Panagiotis Ntostis
Organization name University of Leeds
Department Medicine and Health
Lab LICAMM
Street address Clarendon Way
City Leeds
ZIP/Postal code LS2 9JT
Country United Kingdom
 
Platform ID GPL21290
Series (1)
GSE133592 The impact of maternal age on trophectoderm gene expression profiles in human.
Relations
BioSample SAMN12172582
SRA SRX6385325

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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