RNA sequencing using massive analysis of cDNA ends (MACE)

Total RNA was isolated from formalin‐fixed and paraffin‐embedded (FFPE) sections of all specimens using the Quick‐RNA FFPE Kit (Zymo Research, USA). Following a DNAse I digestion using the Baseline‐ZERO kit (Epicentre, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, USA). The fragment size of all RNA samples ranged between 120 and 150 bp. The preparation of massive analysis of cDNA ends (MACE) libraries was carried out using 1 µg of total RNA, as previously described (Zajac et al, 2015). The barcoded libraries (four CNV membranes and four control samples) were sequenced simultaneously on the NextSeq 500 (Illumina, USA) with 1 × 75 bp. Illumina sequence reads and were processed with an in‐house analysis pipeline (GeneXPro, Germany), including TrueQuant PCR bias elimination and mapping against the human reference genome. For MACE analysis, normalization and test for differential gene expression were calculated using the DEGseq (Wang et al, 2010). Differential gene expression was quantified as the log₂ ratio of the normalized values between the two libraries (CNV vs. control). The sequencing data reported in the current article have been deposited in the Gene Expression Omnibus database under the accession number {"type":"entrez-geo","attrs":{"text":"GSE146887","term_id":"146887"}}GSE146887 (Schlecht et al, 2020).