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Sample GSM7665469

Query DataSets for GSM7665469

Status

Public on Aug 01, 2023

Title

raw_C57_20141436_E15.5 (Run 2)

Sample type

SRA

Source name

neuro retina

Organism

Mus musculus

Characteristics

tissue: neuro retina
genetic background: C57BL/6JRccHsd
cell type: retinal cells
genotype: wild type
developmental stage: E15.5

Treatment protocol

Total of 50 Samples over two separate Runs (Run1/Run2). Two outliers were removed. Input RNA had RIN>8.

Growth protocol

All mice used were maintained on a 99.9% C57BL/6JOlaHsd genetic background with a 12 h day-night cycle and supplied with food and water ad libitum. Crb1KO (Crb1-/-) mice (van de Pavert et al., 2004) were crossed with a retinal Crb2ΔRPC (Crb2F/FChx10CreTg/+ clone P1E9) mouse strain (Alves et al., 2013b; Rowan and Cepko, 2004) to ablate both Crb1 and Crb2 during retinal development from retinal progenitor cells. Cre starts to be expressed around E9.5 in the retina (Rowan and Cepko, 2004). The previously made Chx10Cre mice were generated by knocking in GFP-Cre-IRES-AP cDNA in exon-1 of the Chx10 gene on a Bacterial Artificial Chromosomes (BAC; -55 kb Chx10 of ATG to +22 kb off of the polyadenylation sequence) (Rowan and Cepko, 2004). Interestingly, the last exon of the Abcd4 gene is only ~7000 bp from the last exon of the Chx10 gene thus was on the BAC. Others have shown that large BACS containing several genes can lead to overexpression of flanking genes (Kolisnyk et al., 2013; Ting and Feng, 2014). We believe the integration of the cDNA in close proximity to the Abcd4 gene and being both on the BAC may have led to the overexpression of the Abcd4 gene in Crb1KOCrb2ΔRPC mice. Crb1KOCrb2ΔRPC mice were compared to littermate Crb1KOCrb2F/F mice not expressing Cre. C57BL/6JOlaHsd mice have a 365 kb deletion ablating the Mmrn1 and Snca and express low levels of Abcd4 (Gajović et al., 2006; Specht and Schoepfer, 2004). The C57BL/6J substrain (from the Jackson Laboratory) carries a 5-exon-spanning deletion in the Nnt gene but C57BL/6JOlaHsd have a wildtype Nnt gene (Huang et al., 2006).

Extracted molecule

total RNA

Extraction protocol

Ovation RNA-Seq System V2 (NuGEN) for cDNA amplificationV2.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB 5500 Genetic Analyzer

Description

resultsPeteQuinn_Run2_Tg_vs_C57_V2.txt

Data processing

•Performed sequencing using Life Technologies SOLiD5500, single end, 50 bp. Reads aligned against mm10, bamgen.mqv.threshold set to 20 (Lifescope v2.5)
•Genes with no counts in any of the samples were removed. Filtering: Genes with more than 1 count-per-million reads (CPM) in 5 or more of the samples were kept. Normalization: TMM (edgeR), weighted trimmed mean of M-values (to the reference) is used (Robinson & Oshlack, 2010).
•Original gene symbols mapped to other gene identifiers using biomaRt, using Ensembl (v90). The count data was transformed to log2-counts per million (logCPM) using voom, estimating the mean-variance relationship.
•Benjamini-Hochberg false discovery rate to correct for multiple testing of the resulting p-values. Analysis was performed using R v3.4.1 and Bioconductor v3.5.
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts and CPMs for each sample

Submission date

Jul 27, 2023

Last update date

Aug 01, 2023

Contact name

Jan Wijnholds

Organization name

Leiden University Medical Center

Department

Ophthalmology

Lab

Wijnholds