title: GSM8282004: RGC, PSCL_3mpi, cell21; Mus musculus; RNA-Seq
library_name: GSM8282004
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.Mouth-pipetting was employed to pick single-labeled RGCs manually. A small volume of the RGC suspensions was transferred through a series of 1% BSA-containing PBS drops until a single targeted RGC was isolated. This RGC was then transferred into 4.2 µl lysis buffer, as per the Smart-seq2 protocol, and stored at -80 °C for subsequent processing.The single-cell 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 27 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. Samples with low cDNA quality or low expression levels of both genes were excluded from downstream library construction. Library construction utilized the TruePrep DNA Library Prep Kit V2 for Illumina (vazyme) and TruePrep Index Kit V2 for Illumina, following the manufacturer's instructions. The constructed library was then sent to Novogene company for sequencing with NovaSeq PE150.