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Sample GSM3517550

Query DataSets for GSM3517550

Status

Public on Dec 17, 2021

Title

MWMSmmKONM2: Normoxia EMP2 KO #2

Sample type

SRA

Source name

Retina/coroid

Organism

Mus musculus

Characteristics

strain: C57BL6J
Sex: Male
age: G17
genotype/variation: EMP2 KO
tissue: Retina/coroid

Treatment protocol

Oxygen-induced retinopathy was produced in the mouse according to standard guidelines published [Connor 2009]. Briefly, pups were designated at P0.5 the morning that they were found to be delivered. Mothers were maintained on the standard rodent chow diet, as above, during gestation and during suckling. Litters were culled to 8 pups, as per protocol recommendations. Mothers were randomly assigned to normoxia conditions or hyperoxia conditions. For those assigned to hyperoxia, on postnatal day 7, nursing mothers and their pups were subjected to hyperoxia (75% oxygen continuously) for 5 days by placing them in an airtight chamber (Biospherix Proox model 360). Conditions including humiditiy, temperature, and the health of the animals were assessed twice daily while in the chamber. On postnatal day 12, the mothers and their pups were removed from the chamber and placed back into room air (21% oxygen).

Growth protocol

EMP2 KO mice bred on a C57BL/6 background were obtained from the laboratory of Dr. Carmen Williams [Williams 2017] and re-derived at UCLA. Wildtype C57BL/6 mice were purchased from the UCLA Division of Laboratory and Animal Medicine colony. All mice were housed in 12:12 hour light-dark cycles with ad libitum access to a standard rodent chow diet (Pico Lab Rodent Diet 20, cat# 5053, Lab Diet, St. Louis, MO) and water.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using the Zymo Direct-Zol RNA MiniPrep Plus (cat# R2070, Irvine, CA) as per the manufacturer’s instructions. Total RNA was quantified and 260/280 ratios determined using Nanodrop (all samples with a 260/280>1.97). 1μg of total RNA was used as starting material for each sample.
RNA sequencing and library preparation was performed by the UCLA Technology Center for Genomics and Bioinformatics with Kapa hyper Prep Kits with RiboErase. The workflow consists of rRNA depletion, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000 for a single read 50 run.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Data processing

Data quality check was done on an Illumina SAV. Demultiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program.
The STAR ultrafast universal RNA-seq aligner (v2.5.2b) was used to generate the genome index and perform single-end alignments. Reads were aligned to a genome index that includes both the genome sequence (GRCm38 primary assembly) and the exon/intron structure of known gene models (Gencode M12 genome annotation). Alignment files were used to generate strand-specific, gene-level count summaries with STAR's built-in gene counter. Differential expression analysis was performed with DESeq2.
Counts were normalized per-sample in units of RPKMs after correcting for gene mappable length and sample total counts.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Tab delimited, per-sample, gene expression estimates in units of RPKMs

Submission date

Dec 17, 2018

Last update date

Dec 17, 2021

Contact name

Madhuri Wadehra

E-mail(s)

mwadehra@mednet.ucla.edu

Phone

310-825-1590

Organization name

UCLA