GSM3186816
GSM3186816: Opn4_Sham_1; Mus musculus; RNA-Seq
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.
GEO
GSM3186816
GEO Accession
GSM3186816