384 well c1000 cycler

A total amount of 1µg of RNA was in-vitro transcribed into cDNA by using 1 µL dNTP-Mix (10 mM), 1 µL Oligo(dT)18 Primer (0.5 µg/µL), 4 µL RT Buffer (5×), 1 µL Maxima H Minus Reverse Transcriptase (200U/µL), and 0.5 µL RiboLock RNase Inhibitor (40U/µL) (all Thermo Fisher Scientific) on a S1000 cycler (BioRad) at 52 °C for 0.5 h. Further, qRT-PCR runs were performed on a 384-well C1000 cycler (BioRad). In general, all samples were analyzed in technical triplicates using 7.34 ng of cDNA for each replicate and the SYBR-green-based Luna Universal qPCR Master Mix (New England Biolabs, Frankfurt am Main, Germany). At the end of each run, melting curve analyses were performed. Oligonucleotide sequences are given in Table 1 (Table 1).

RNA was isolated using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. In vitro transcription or RNA and qRT‐PCR was performed as described previously [26 (link)]. Gene expression was determined on the 384‐well C1000 cycler (BioRad) with oligonucleotides given in Table S2. GAPDH and ACTB were used as housekeeping genes and for data normalization. RNA samples used for transcriptome analyses were assessed as described previously [26 (link), 27 , 34 (link)]. Only RNA with an integrity number of > 8.5 was analyzed. RNA‐sequencing data are freely available via GEO. The data are available via the NCBI Gene Expression Omnibus (GEO) database repository (GSE195794; https://www.ncbi.nlm.nih.gov/geo/).