Transcriptional Profiling of GFP-SOX10 in MN1 Cells

The human SOX10 open-reading frame in pDONR221 (Addgene plasmid #24749) was obtained and SOX10 was cloned into pcDNA-DEST53 using LR Clonase (ThermoFisher) to generate a GFP-SOX10 expression construct. MN1 cells [29 (link)] were plated at a density of 100,000 cells per mL and transfected with the GFP-SOX10 expression construct using Lipofectamine 2000 (ThermoFisher). After 48 h, cells were harvested, suspended in PBS, and sorted into GFP-positive and GFP-negative populations using a SY3200 Cell Sorter (Sony). RNA was isolated from each cell population using the RNeasy Mini Kit (QIAGEN). A CAGE library was generated for each sample as described [47 (link)], with modifications made for compatibility with current generation Illumina sequencing platforms. Each library was sequenced on an Illumina MiSeq (4 million reads generated per library). The sequencing data was analyzed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), filtered using TagDust2 [68 (link)], mapped using BWA [69 (link)], clustered using Paraclu [70 (link)], and visualized using BEDtools [71 (link)] and the UCSC Genome Browser [23 (link)].