Ionxpress plus gdna and amplicon library preparation kit

Sequencing was performed using four to five biological replicates for each condition. RNA was extracted using the Qiagen Micro-Rneasy Kit (Qiagen, catalog no. 74004) according to the manufacturer’s protocol. As external control, 1 μl of ERCC RNA Spike-In Mix (Thermo Fisher Scientific, catalog no. 4456740) diluted at 1:5000 was added to the isolated RNA. cDNA was synthesized with the Ovation RNA-Seq system v2 (NuGEN, catalog no. 7102). As input, 100 ng of cDNA was used for library preparation using the IonXpress plus gDNA and Amplicon library preparation kit (Thermo Fisher Scientific, catalog no. 4471269). To achieve size selection of the library, a 2% E-Gel was used, and the samples were barcoded and subsequently amplified. Further quantification for each sample library was performed using the KAPA Library Quantification Kit (KAPA, catalog no. KK4827). Of each sample, equal amounts were sequenced on an Ion Torrent Sequencer. On the basis of the barcodes, the raw reads (Fastq) were split into sample-specific reads and subsequently checked for sequence quality and sequence repeats. Low-quality bases and short reads were trimmed or excluded from further analysis. The reads were mapped to Mus musculus genome (mm10) using TMAP Aligner and quantified using Partek Flow software. Genes had to have at least five reads in at least 80% of the samples to be considered for further analysis.