Human clariom s pico arrays

Sorted cell populations were snap frozen in PBS directly after sorting and stored at –80°C until RNA extraction. QIAzol Lysis Reagent (Qiagen) was added to the cells, and RNA was isolated and purified using the RNeasy kit (Qiagen). The RNA concentration was measured on a NanoDrop ND‐2000 (Thermo Scientific) and the RNA integrity was determined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was amplified using the GeneChip WTF 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 by the GeneChip Fluidics Station 450 and the 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 sing the RMA algorithm implemented in the limma Bioconductor R‐Package [42]. Adjusted P‐values were calculated using the Benjamin‐Hochberg method. Data were visualized using glimma and complex heatmap R‐packages [43]. The following packages were used in the analysis pipeline: oligo, limma, affycoretools, clariomshumantranscriptcluster.db, GO.db, Glimma, biobroom, ComplexHeatmap, pathfindR, ggbiplot, tidyverse [42, 43, 44, 45, 46, 47, 48].