Single-cell RNA-seq of tissue-adjacent cells

Cells on and in the region adjacent to the 350 μm diameter electrodes were manually dissected using a 1.5 mm biopsy punch (Integra LifeSciences). RNA was harvested using NucleoSpin RNA XS Kit (Macherey-Nagel) and converted into cDNA using SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories). DNA libraries were prepared with Ion Xpress™ Plus gDNA Fragment Library Preparation kit and sequenced by an Ion Proton next-generation sequencer (Thermo Fisher Scientific Inc.). The resulting sequences were aligned to the human transcriptome and genome (hg38) using a two-stage alignment pipeline employing STAR and TMAP read aligners [36 (link)]. The number of reads per protein-coding mRNA was determined using Partek Genomics Suite (Partek Inc.), and the dataset was normalized using the trimmed mean of the M-values method [37 (link)]. Genes with reads per million (RPM) ≥1 in three or more samples were selected (S1 Table), and differential expression and statistical analysis were carried out using the classic implementation of edgeR (S2 Table) [38 (link)]. The RNA-Seq data and methods can be accessed through the Gene Expression Omnibus (GEO: GSE146884).