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Sample GSM1643940

Query DataSets for GSM1643940

Status

Public on Mar 26, 2015

Title

VC_KO_1

Sample type

SRA

Source name

Visual Cortex, 8 weeks of age

Organism

Mus musculus

Characteristics

genotype: MeCP2 KO
tissue: visual cortex

Treatment protocol

Mice were housed under standard conditions in the animal facility and dissected at the same time point in the light-dark cycle for each dissection.

Extracted molecule

total RNA

Extraction protocol

Visual Cortex was dissected from 8-10 week old MeCP2 knockout mice (Mecp2tm1.1Bird) or wild-type littermate controls. RNA was trizol and extracted using Qiagen Rneasy purification with on column DNase steps. ERCC control RNAs were spiked into the samples with equal mass added per visual cortex (note: they were not used in subsequent analysis).
Illumina library construction and sequencing was carried out as follows: total RNA was depleted of ribosomal RNA using the Ribozero rRNA removal kit (Epicentre), heat-fragmented to 200-700 bp in length and cloned using Uricil-N-Glycosylase-based strand-specific cloning. cDNA fragments were sequenced using an Illumina HiSeq 2000
Strand-specific, rRNA depleted total RNA, Single-end 49 bp sequencing

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Total RNA was depleted of rRNA by hybridization (using RiboZero) and heat fragmented. Strand-specific RNA-Seq libraries were prepared using the dUTP method, and sequenced on an Illlumina Hiseq instrument

Data processing

Base calling was performed using standard approaches.
Illumina raw reads in .fastq files were aligned using bwa 0.6.2. Target sequences included (1) mm9 autosomal and sex chromosomes plus (2) a read-length-dependent splice library (~3M sequences, each 50-98bp). The latter comprised all possible minimal intragenic sequences of two or more exons (based on the RefSeq annotation) for which one or more exon-exon junctions could be crossed by reads of the required length. After bwa-indexing the targets with the bwtsw algorithm, reads were aligned with zero trimming, zero gaps, and allowing up to 5 mismatches. The sam/bam files output by bwa were indexed and sorted and served as input for further processing.
Aligned reads were minimally filtered for QC, removing any reads with uncalled bases or with unmapped or nonuniquely mapped sequences.
Employing "MAPtoFeatures" perl scripts, the loci of uniquely mapped reads were compared to the loci of all genic features (exons, introns, UTRs, CDSs, etc., and all junctions between them) of (1) all genes based on the RefSeq annotation and (2) all rRNA genes based on RepeatMasker, for the alignment target genome. Average exon Density (read coverage per bp) was calculated, equal to rdbp per feature length. Each sample's Densities were renormalized from total reads not in noncoding genes and not in rRNA to a standard total of 10M reads, and to a standard read length of 35bp. With these conventions, units of Density always equal RPKM times 0.35. Quantile normalized exon densities were treated as expression levels comparable among all samples.
Genome_build: For all samples mm9 (NCBI37, Jul7. 2007).
Supplementary_files_format_and_content: Entrez gene ID, refseq gene name, gene length, and exon densities are listed for each of the 14168 genes analyzed across expression platforms in this study.

Submission date

Mar 25, 2015

Last update date

May 15, 2019

Contact name

Harrison Wren Gabel

E-mail(s)

gabelh@pcg.wustl.edu

Organization name

Harvard Medical School

Department

Neurbiology