RNA sequencing of cell populations isolated from one GW14.5 human fetal cortex

To understand prenatal cortex on transcriptional level, we profiled the transcriptomes of ventricular radial glial cells (vRGs), outer RGs (oRGs), intermediate progenitors (IPs) and neurons sorted from one GW14.5 prefrontal cortex. Prefrontal cortex tissues were processed by mechanical cutting and trypsin digestion to obtain single-cell suspention. Cells were fixed and incubated with primary antibodies: anti-CRYAB (Abcam, ab13496), anti-TBR2 (R&D systems, AF6166) , anti-HOPX (Sigma, HPA030180), and anti-SATB2 (Abcam, ab69995), followed by incubation of secondary antibodies conjugated with Alexa-488 or Alexa-555. Neural cells sorting used acknowledged marker: CRYAB+TBR2- cells as vRGs; HOPX+CRYAB- cells as oRGs; TBR2+ cells as IPs; SATB2+TBR2- cells as neurons. Total RNA of each cell population was extracted by miRNeasy FFPE Kit (Qiagen) following the manufacture’s protocol. Fusion transcripts were recognized by SOAPfuse software. Differentially expressed genes among each cell type were identified using the R package Ballgown

为从转录水平解析产前大脑皮层，我们对1例孕14.5周（GW14.5）的前额叶皮层中分选得到的室管膜放射状胶质细胞（ventricular radial glial cells, vRGs）、外侧放射状胶质细胞（outer RGs, oRGs）、中间祖细胞（intermediate progenitors, IPs）以及神经元的转录组进行了表征。前额叶皮层组织通过机械解离与胰酶消化制备为单细胞悬液。细胞经固定后，依次与一抗进行孵育：抗CRYAB抗体（Abcam，ab13496）、抗TBR2抗体（R&D Systems，AF6166）、抗HOPX抗体（Sigma，HPA030180）以及抗SATB2抗体（Abcam，ab69995），随后再与标记有Alexa-488或Alexa-555的二抗进行孵育。神经细胞分选采用经过验证的分选标志物组合：以CRYAB+TBR2-细胞作为vRGs，HOPX+CRYAB-细胞作为oRGs，TBR2+细胞作为IPs，SATB2+TBR2-细胞作为神经元。按照试剂盒制造商的实验流程，使用miRNeasy FFPE试剂盒（Qiagen）提取各细胞群的总RNA。通过SOAPfuse软件识别融合转录本。使用R语言包Ballgown鉴定不同细胞类型间的差异表达基因。