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Sample GSM3912682

Query DataSets for GSM3912682

Status

Public on Jul 20, 2021

Title

BRP233R

Sample type

SRA

Source name

middle

Organism

Homo sapiens

Characteristics

tissue: Trophectoderm
developmental stage: Blastocyst
day of development: Day 5
maternal age: between 30 and 39 years

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