Supplementary Table 1_Sterken et al 2021.xlsx

RNA was isolated from three pools of BT-20 WT cells and three BT-20 CEBPB-ko clones, using the RNeasy Plus Mini Kit (QIAGEN). Quality and quantity of RNA was determined using Agilent’s Bioanalyzer 2100 in combination with the RNA 6000 nano chip (Agilent). Library preparation was done using Illumina’s TruSeq RNA v2 kit following the manufacturer’s description. The libraries were quality checked and quantified using Bioanalyzer 2100 in combination with the DNA 7500 kit (Agilent). Sequencing was done on a HiSeq2500 in 50 bp, single-end sequencing, high-output mode. Sequence information was extracted using bcl2fastq v1.8.3 (Illumina), Sequencing resulted in around 50 million reads per sample. Quality of the reads was checked using FastQC (v. 0.11) and filtering and trimming of low-quality reads was performed using Trimmomatic (v. 0.33). Trimmed reads were aligned to the GRCh38/mm10 genome (genome annotation from ensemble release 92) using STAR aligner v. 2.6.0b60. Identification of differentially expressed genes (DEGs) was done using the R package DESeq2 27. The resulting p-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate61. Genes were regarded as differentially expressed if adjusted p-values were < 0.05. Functional clustering analysis was performed using the DAVID database (https://david.ncifcrf.gov/, version 6.7) at default settings with medium stringency (downregulated genes figure 2d) or highest stringency (figure S1A). GSEA was performed using the GSEA desktop application and compared to the Hallmark gene sets. The phenotype label was set as CEBPB WT vs CEBPB-ko. The t-statistic mean of the genes was computed for each hallmark gene set using a permutation test with 1000 replications.