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Sample GSM1816961

Query DataSets for GSM1816961

Status

Public on Jan 01, 2016

Title

NE1468

Sample type

SRA

Source name

Adult Neocortex tissue

Organism

Mus musculus

Characteristics

brain region: Neocortex
Stage: PC14
strain: C57BL/6J
Sex: Female

Treatment protocol

Nulliparous B6 females were trio bred to B6 males to generate timed pregnancies. Post-conception (PC) day 1 is the day the vaginal plug was observed. Postpartum samples are abbreviated PP. Randomly cycling, age-matched virgin females were used to establish the basal level of gene expression to compare to PC14, PC16, PP1, PP3 and PP10 dams.

Growth protocol

After eight-week-old male and female C57BL/6J (B6) mice were purchased from the Jackson Laboratory (Bar Harbor, ME), nulliparous B6 females were trio bred to B6 males to generate timed pregnancies. The breeding condition is repeated in B99. Mice were fed standard chow diet (Purina 5001) and distilled water ad libitum.

Extracted molecule

total RNA

Extraction protocol

The cerebellum, hippocampus, hypothalamus, and neocortex were dissected rapidly, flash-frozen in liquid nitrogen, and placed on dry ice until storage at -80°C. Frozen brain tissue was homogenized in 1ml TRIzol® solution (Life Technologies™ 15596-018), using a motorized homogenizer. Samples were than centrifuged at 4°C for ten minutes at 4,000 rpm to separate out debris and the resulting supernatant was processed for total RNA extraction procedure as manufacture suggested.
Total RNA was treated with TURBO™ DNase (Ambion® AM223) and purified with Zymo Research RNA Clean & Concentrator™-25kit (Zymo Research R1018). 5ug (cerebellum, neocortex, and hippocampus) or 1ug (hypothalamus) of total RNA was used for subsequent steps. The External RNA Controls Consortium (ERCC) RNA Spike-in Controls (Ambion® 4456740) 1:10 and 1:100 were added to the total RNA 5ug and 1ug, respectively as suggested. NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs® Inc. E7530L), 12 PCR cycles, quality was examined using Agilent High Sensitivity DNA Bioanalyzer Chips (Agilent Technologies 5067-4626) and quantified by KAPA Library Quantification Kits for Illumina sequencing platforms (KAPA Biosystems KK4824).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

NE1468_GCCAAT_R1

Data processing

Sequences from Illumina adapters were trimmed using Trimmomatic ( version 0.32, minlen set to 50, 2 seed mismatches )
Unique reads were aligned to Mus musculus genome, release 75 (Ensembl) using Tophat ( version 2.0.8, --read-mismatches and --read-edit-dist set to 6 )
Expression was quantified for genes, to raw counts using easyRNASeq ( from Bioconductor version 3.0.2, geneModels summarization )
edgeR, Fisher's exact test for differential expression ( from Bioconductor version 3.0.2, tagwise dispersion, Benjamini-Hochberg FDR correction )
Cufflinks quantification to Fragments Per Kilobase Per Million Mapped Reads (FPKM) (Cufflinks version 2.1.1, -- no-effective-length-correction )
Genome_build: Ensembl release 75
Supplementary_files_format_and_content: Excel file containing raw count information
Supplementary_files_format_and_content: Excel file containing FPKM information

Submission date

Jul 10, 2015

Last update date

May 15, 2019

Contact name

Michelle Arbeitman

E-mail(s)

michelle.arbeitman@med.fsu.edu

Phone

850-645-9846

Organization name

Florida State University