Transcription factor network analysis identifies REST/NRSF as an intrinsic regulator of CNS regeneration

The goal of this study is to use RNA-seq to investigate the molecular mechanisms by which REST inhibition regulates corticalspinal axon regeneration. To achieve this goal, cortical motor neurons from adult wild-type (WT) and REST conditional knockout (cKO) were purified at 1, 3, 7 days following spinal cord injury (SCI) or sham operation. RNA-seq libraries for cortical motor neurons were prepared with the QuantSeq mRNA-Seq library prep kit FWD for Illumina (Lexogen) and sequenced using HiSeq4000 (Illumina). Sequenced reads were mapped to reference genome (mm10) using STAR, and count level data were quantifed using Salmon. Differential gene expression analysis was performed using limma voom. Overall design: mRNA profiles were performed in cortical motor neurons from wild-type or REST cKO mice that were recovered 1, 3, 7 days following SCI or sham operation. Each group has 3-4 replicates with ~1000 cells in each replicate.

本研究旨在通过RNA测序（RNA-seq）探究REST抑制调控皮层脊髓轴突再生的分子机制。为达成该研究目标，我们于脊髓损伤（SCI）或假手术后的第1、3、7天，从成年野生型（WT）及REST条件性敲除（cKO）小鼠体内纯化皮层运动神经元。针对皮层运动神经元的RNA测序文库，采用Lexogen公司的Illumina适配QuantSeq mRNA-Seq文库制备试剂盒FWD进行构建，并通过Illumina HiSeq4000平台完成测序。测序reads通过STAR软件比对至参考基因组mm10，基因计数数据经Salmon软件完成定量。差异基因表达分析采用limma voom方法开展。实验整体设计：对脊髓损伤或假手术后恢复1、3、7天的野生型或REST条件性敲除小鼠的皮层运动神经元进行mRNA表达谱检测；每组设置3-4个生物学重复，每个重复约包含1000个细胞。