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Sample GSM3186821

Query DataSets for GSM3186821

Status

Public on May 29, 2019

Title

Opn4_3dpc_3

Sample type

SRA

Source name

Retina

Organism

Mus musculus

Characteristics

genotype: Opn4CreERT/+;R26-tdTomatof/f
tissue: retina
cell type: retinal ganglion cell

Extracted molecule

polyA RNA

Extraction protocol

Tissue Preparation: Opn4CreERT/+;R26-tdTomatof/f (Opn4) and HB9:GFP (HB9) mice were used for the RNA sequencing experiment. HB9:GFP mice were also injected with tamoxifen. Tamoxifen was injected I.P. (0.124mg/g body weight) for 5 consecutive days starting at P21. At P42 animals underwent optic nerve crush (Crush) or sham surgery 3 days prior to tissue collection. Adult mouse RGCs were dissociated. The retina was removed in HBSS (ThermoFisher, 14170161) and transferred to DMEM (ThermoFisher, 11995065) and 17U/ml papain (Worthington Biochem, PAPL). The retina was enzymatically dissociated for 30 minutes at 37°C in a water bath. Following incubation with papain, the retina was washed 5 times with DMEM. The retina was then triturated in FluoroBrite DMEM (ThermoFisher, A1896701) with B27 supplement and 20mM HEPES (ThermoFisher, 15630080). The cells were briefly spun down, resuspended in FluoroBrite DMEM + B27 + HEPES, and passed through a 100μm filter. Cells were plated at low density and allowed to settle. Under epifluorescent illumination tdTomato+ or GFP+, RGCs were located. Phase contrast was used to visualize that the cell was isolated from other cells. Pipettes were pulled with a Sutter Instrument puller. Tips were trimmed to the desired size. A Sutter Instrument micromanipulator was loaded with a pipette with an opening slightly larger than the cell to be captured. Care was taken to prevent excess media from entering the pipette as it was positioned in the media. A cell was only collected if it could be picked up without any additional contaminating cells. The collected cell was promptly lysed in RNA lysis buffer. Cells were only collected for up to 2 hours after the animal was euthanized. 10 cells were collected from each animal. 3 pools of 10 cells were combined to make 1 biological sample.
RNA-purification for sequencing: The Absolutely RNA Nanoprep kit (Agilent, 400753) was used to purify RNA. RNA was purified according to the manufacturer’s protocol. 70% ethanol was substituted for 80% sulfolane. RNA was eluted in 11μl of 1:1 elution buffer:water. RNA was reverse transcribed and pre-amplified for 11 cycles with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, 634888) according to the manufacturer’s protocol. An aliquot of the pre-amplified product was reserved for quality control qPCR.
Ocean Ridge Biosciences (Deerfield Beach, FL) sequenced the RNA. Briefly, cDNA was assessed for quality by capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA chips (Agilent Technologies, Santa Clara, CA). 100 pg of pre-amplified cDNA was used for library preparation. The Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA) was used to prepare cDNA libraries for sequencing. The libraries were pooled at equimolar concentrations and sequenced on an Illumina HiSeq Flow Cell v4 with 50-bp paired-end reads. All samples had a minimum of 30 million passed-filter 50nt paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

SL142658
pre-amplified mRNA, tRNA depleted

Data processing

Reads were aligned using TopHat v2.0.11 to the mm10 Bowtie genome index.
Reads were counted using the Bioconductor package easyRNASeq v1.6.0.
edgeR v3.14.0 was used to calculate differential gene expression.
Genes were included if they were expressed at ≥ 1 count per million in at least 3 of the 12 samples. Genes were considered differentially expressed if log2FC ≥ 1 and the FDR adjusted p-value ≤ 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file including annotations and RPKM values for each Sample

Submission date

Jun 12, 2018

Last update date

May 29, 2019

Contact name

Kyung (Kevin) Park

E-mail(s)

kpark@miami.edu

Phone

3052432493

Organization name

University of Miami