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Sample GSM1273824

Query DataSets for GSM1273824

Status

Public on Nov 23, 2013

Title

AY4

Sample type

SRA

Source name

semen

Organism

Homo sapiens

Characteristics

tissue: whole semen
phenotype: Normozoospermic
molecule subtype: rRNA depleted total RNA

Extracted molecule

total RNA

Extraction protocol

For improved total RNA isolation, the ejaculate was incubated at 37⁰C or immediately washed with an equal volume of Sperm Washing Medium, at 37⁰C (SWM, Irvine, Santa Ana, CA). SWM contains 21 mM HEPES buffer, 4 mM Sodium Bicarbonate, and 5 mg/mL Bovine Serum Albumin. The cell mixture was spun at 680 x g for 12 minutes. Supernatant was removed and the sperm pellet was resuspended in the residual liquid. The samples were subjected to minimal mixing and spinning in order to preserve RNA. Cell suspensions were completely homogenized by vigorous vortexing and used for RNA isolation with warmed (37⁰C) TRIzol reagent (Life Technologies, Carlsbad, CA). RNA was isolated according to the manufacturer (Invitrogen) and the RNA pellet was resuspended in RNase-free water. Total RNA was DNase treated and purified using an RNeasy Mini Kit spin column (Qiagen) and diluted in RNase-free water.
Ribosomal RNA reduction was carried out via the RiboMinus Eukaryote Kit for RNA-Seq (Life Technologies). 500 ng of ribosomal-free RNA and random primers were used for whole transcriptome library preparation in total RNA-Seq protocol (Life Technologies). Final libraries were quantified using qPCR NGS Library Quantification Kit (AB SOLiD, Life Technologies). Equimolar amounts of each cDNA library were pooled and ePCR was performed using the EZBead system (Life Technologies). Enriched templated beads were applied to AB SOLiD flow cell and 75 bp sequencing reads were collected using the Solid 5500xl sequencer (Life Technologies).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB 5500xl Genetic Analyzer

Description

Sample 1

Data processing

Reads from each sample were mapped using Tophat 2.0.6 mapper with Bowtie aligner (Langmead et al. , 2009, Trapnell et al. , 2009) to the most updated reference sequence (hg19).
Expression representation was determined as length-based average coverage values using our in-house software RNASeqnator (Sethi & Lyons-Weiler, personal communication). RNASeqnator renders a ‘gene-level’ analysis for all exons reported under any NCBI identifier to co-occur in an mRNA transcript. This is conducted as a ‘superset’, and we therefore refer to that measurement as a “supertranscript”, or ST, analysis. ST measurements were compared between groups using methods selected by Efficiency Analysis (Jordan et al. , 2008). Efficiency analysis points to transformation and normalization methods, and to tests for difference, that are optimally reproducible using a re-sampling scheme.
Genome_build: hg19
Supplementary_files_format_and_content: excel file containing cube root normalized coverage; Efficiency analysis indicated that median/mean normalization followed by cube root transformation was highly reproducible (internally consistent) compared to other methods for studying differential expression.

Submission date

Nov 22, 2013

Last update date

May 15, 2019

Contact name

Andrew Georgiadis

E-mail(s)

georgiadisa@upmc.edu

Organization name

UPMC

Street address

203 Craft Ave.