title: Single-cell trajectory analysis of the differentiation of RGCs in the VZ (scRNA-seq)
design: Single-cell trajectory analysis of the differentiation of RGCs in the VZ (scRNA-seq)
library_construction_protocol: Dissection of VZs from neonatal mouse brains was performed as described 1. Briefly, the midbrain, the olfactory bulb, meninges and the hippocampus were removed in sequence to attain the telencephalon. Then VZ-containing tissues were separated from the telencephalon. Tissues from 4 mice were pooled, transferred to a 1.5ml Eppendorf tube, and digested with freshly prepared 10U/ml Papain solution (Worthington, catalog no. LS003126) at 37Â°C for 30 minutes. Then the Papain solution was aspirated, and 1ml culture medium (20% FBS) was added, followed by 1 minute of incubation to terminate the digestion. The supernatant was removed, and another 1ml culture medium (20% FBS) was added. The digested tissues were gently pipetted up and down by 20 times with a P1000 tip to dissociate the cells mechanically. Centrifuge at 350rcf for 5 minutes and discard the supernatant. DNA was purified first by Qiagen minElute PCR purification kit according to the manufacturer's instruction, then by AmupureXP beads with 0.5X upper cutoff and a 1.2X lower cutoff according to manufacturer's instruction. Libraries were constructed using the 10x Genomics Gene Expression 3' kit according to the manual.