Ion proton next generation sequencer

To enrich for the non-ribosomal RNA transcriptome, RNA samples were first depleted of ribosomal RNA using the Low Input Ribominus Kit (Life Technologies). External RNA Controls Consortium (ERCC) spike-in controls (Pool 1) were introduced for all samples (Life Technologies). RNA libraries were constructed using the Ion Total RNA-Seq Kit for AB Library Builder System (Life Technologies) per manufacturer’s instructions, except for a single modification where enzymatic shearing of the RNA was performed only for 15 s due to the already fragmented nature of RNA derived from FFPE. Libraries were barcoded using the IonXpress RNA-Seq Barcode 1-16 Kit (Life Technologies), and libraries were quantified using the Agilent TapeStation 2200 along with the DNA D1K Kit. Libraries were diluted to a concentration of 11 pM prior to templating and emulsion PCR using the Ion Template OT2 200 v2 kit along with Ion OneTouch 2 instrument (Life Technologies). Templates were quantified using the IonSphere Quality Control Kit (Life Technologies). Samples were sequenced on an Ion Proton Next-Generation Sequencer using the Ion Proton PI chip and the Ion PI Sequencing 200 v2 kit (Life Technologies). Samples were sequenced using two RNA-Seq libraries/samples per chip to an average of 30–40 million reads per sample. RNA sequencing data is to be submitted to Gene Expression Omnibus.

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).

TCR-seq libraries for next generation sequencing (NGS) were prepared from the mRNA of sorted T cell samples. Details of the modified procedure have been described in the Supplementary Material. In short, total mRNA was converted to cDNA using reverse transcriptase, and universal sequences for unbiased PCR amplification were added to the 5′ end by poly-A tailing with the terminal deoxynucleotidyl transferase reaction and subsequent second strand synthesis using a universal primer. The TCR locus was amplified by nested PCR with the universal primer and the TCR constant region-specific primers. Amplified TCR libraries were then fragmented enzymatically and sequencing adaptors and barcodes were added to the TCR libraries using ligation and subsequent PCR. Final TCR libraries with 200–300 base pairs were sequenced using an Ion Proton next generation sequencer (Thermo Fisher Scientific). The raw data from these experiments have been deposited at the NCBI GEO; accession GSE115425. The sequences of primers and adapters are shown in Supplementary Table 1.