Profiling Transcriptome of Sorted Cells

To isolate total RNA, sorted cells were flash frozen in PBS immediately after sorting and stored at −80°C before RNA extraction. QIAzol Lysis Reagent (Qiagen) was added to the cells, and RNA was isolated and purified using the RNeasy kit (Qiagen). The concentration was measured on a NanoDrop ND-2000 (Thermo Fisher Scientific), and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was amplified using the GeneChip WT Pico Kit (Thermo Fisher Scientific) generating biotinylated sense-strand DNA targets. The labeled samples were hybridized to human Clariom S pico arrays (Thermo Fisher Scientific). Washing and staining was performed using the GeneChip Fluidics Station 450, and scanning was performed using the GeneChip Scanner 3000 (both Thermo Fisher Scientific). All cell populations were generated in triplicate. All data analysis was performed in RStudio. Raw data were normalized using the robust multi-array average (RMA) algorithm implemented in the limma Bioconductor R-package (Ritchie et al., 2015 (link)). Adjusted P values were calculated using the Benjamini–Hochberg method. Data were visualized using glimma and pheatmap R packages (Su et al., 2017 (link)). The R package UpsetR was used to visualize cloning experiments (Lex et al., 2014 (link)).