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Sample GSM3926693

Query DataSets for GSM3926693

Status

Public on Jul 04, 2019

Title

Testis, 1/6612 cre+ E2F3 lox/wt Rb lox/lox

Sample type

SRA

Source name

testis

Organism

Mus musculus

Characteristics

age: 2.5mo
strain: mix: B6, C57BL/6, FVB
age: 2.5mo
genotype: SCRbKOE2F3+/-
tissue: testis

Extracted molecule

total RNA

Extraction protocol

Testes were removed, flash frozen on dry ice and total RNA was isolated from P10 (control only) and adult mouse testis (n=4-5/group) using the RNeasy Mini kit (cat. # 74104, Qiagen). Subsequently, the obtained total RNA was treated with RNase-free DNase (cat. # 79254, Qiagen) and purified using the RNeasy MinElute Clean-up kit (cat. # 74204, Qiagen). RNA quality and concentration were determined using Nanodrop.
The library preparation and sequencing were performed at the FFGC (Finnish Functional Genomics Centre, Turku, Finland). Prior to the library preparation the quality of the total RNA samples was ensured using Agilent Bioanalyzer 2100 and Advanced Analytical Fragment Analyzer. Total RNA samples were pure, intact and all samples had similar quality. Bioanalyzer RIN values were > 9.0. Fifty ng of RNA was taken for the library preparation. Library preparation was done according to Illumina TruSeq® Targeted RNA Expression Guide (part # 15034665).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

2.5 months SC_RB_E2F3_NormalizedHitsPerSample_M1
RNA30

Data processing

The 44 libraries were pooled into a single pool
The samples were sequenced in a single run with Illumina MiSeq instrument using v2 sequencing chemistry.
Single-read sequencing with 1 x 50 bp read length was used, followed by 6 bp + 8 bp dual index run.
Technical quality of the MiSeq run was good and the cluster amount was as expected. Greater than 90% of all bases above Q30 was requested.
The results were submitted to the Illumina BaseSpace data-management and analysis interphase.
The TruSeq Targeted RNA sequencing application v1.0 was used to analyze differential expression of the transcripts in the experimental groups. Briefly, the depth of sequencing between samples was estimated and the expression level for each replicate were normalized based on the total number of aligned reads. The normalized transcript abundance was modeled by a negative binomial distribution model and this model was used to derive a p-value for the differential expression of each transcript. Finally, the application corrected for multiple hypothesis testing by adjusting the p-value for false discovery rate (FDR) by using the Benjamini-Hochberg method.
Genome_build: Hprt, Rpl19 and Ppia were used as reference genes for normalization during the analysis.
Supplementary_files_format_and_content: The TruSeq Targeted RNA sequencing application v1.0 was used to analyze differential expression of the transcripts in the experimental groups.

Submission date

Jul 03, 2019

Last update date

Jul 07, 2019

Contact name

Emmi Rotgers

E-mail(s)

[email protected]

Organization name

University of Turku

Department

Institute of Biomedicine