title: GSM8152428: RGCOlig2_2; Mus musculus; RNA-Seq
library_construction_protocol: For the isolation of single RGCs, magnetic beads coupled with CD90.2 antibody, which specifically recognizes RGC cells, were employed. The beads-isolated GFP-positive RGCs were manually selected for RNA-seq analysis. All RGCs settled in a 35 mm dish for 5 minutes, and subsequently, 10 ~ 15 RGCs were collected into the RNA lysate buffer (SMART-Seq® HT Kit) using UnipicK (DCU ID = 20 μm) under an inverted fluorescence microscope (Olympus). Whole transcriptome amplification in PCR tubes were performed following SMART-Seq® HT Kit protocol. Briefly, 10~20 picked cells (~0.2μl) were lysed in 10μl lysis buffer (with RNase Inhibitor). Next, samples were mixed well with 1μl 3'-SMART-Seq CDS Primer II A, and then incubated at 72°C for 3 minutes and immediately placed on ice for 2 minutes. Then, we incubated samples with 12.5μl One-Step Master Mix at 42°C for 90 minutes and amplified for 20 PCR cycles. The cells' cDNA was purified by immobilization on AMPure XP beads and cDNA is eluted with 17μl Elution Buffer. After purification, 1μl the profile of the cDNA was evaluated using the Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).