title: GSM8282042: RGC, OTI3D, rep1-2; Mus musculus; RNA-Seq
library_name: GSM8282042
library_construction_protocol: The RGC-labeled retina was quickly dissected out from mice after cervical dislocation and incubated with 0.5 mg/ml papain at 37 °C for 30 minutes. The retina was then washed with Neurobasal-A medium and triturated into a single-cell suspension by pipetting. After centrifugation at 300g for 6 minutes at 4 °C, the retinal cells were resuspended in Neurobasal-A medium supplemented with 10% B27.Fluorescence-activated cell sorting (FACS) was used to isolate labeled RGCs. AAV-DIO-GFP was injected into the vitreous body of vGlut2-cre mice eye to label RGCs one month before any injury was applied to the animals. FACS gating was performed to isolate the desired population by sequentially excluding dead cells (DAPI-negative), selecting singlet cells, and then gating on GFP-positive cells to enrich the GFP-expressing population. 200 cells from one eye were collected into one tube containing smart-seq2 lysis buffer as a biological replicate. The RGC lysate underwent further processing according to the Smart-seq2 protocol. RNA was reversely transcribed into cDNA using superscript II and TSO primer and followed by amplification with KAPA HiFi HotStart ReadyMix and ISPCR primer for 19 cycles. The amplified product was purified using Ampure XP beads. A fragment analyzer assessed cDNA quality and Qubit dsDNA High Sensitivity (HS) kit measured cDNA concentration. qPCR measured the relative expression level of the Sncg and Rbpms genes. Then the RGC cDNA was sent to BioCRF in HKUST for full Next Generation Sequencing (NGS) service.