247 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 Patient RNA sequencing for UPR genes. Human cardiac tissue collection was approved by the Biobank Research Ethics Committee, University Medical Center Utrecht, Utrecht, the Netherlands (protocol number WARB 12/387). Written informed consent was obtained or in certain cases waived by the ethics committee when obtaining informed consent was not possible due to the death of the individual. Heart samples collected at autopsy or transplantation were obtained from a homogeneous cohort of patients who all carried the same pathogenic PLN R14del mutation (n = 6). Four control hearts obtained from rejected organ donors (n = 3) or from autopsy (n =1) were used as a reference. RNA was isolated using ISOLATE II RNA Mini Kit (Bioline) according to the manufacturers’ instructions with minor adjustments. After the selection of mRNA, libraries were prepared using the NEXTflexTM Rapid RNA-seq Kit (Bio Scientific). Libraries were sequenced on the Nextseq500 platform (Illumina), producing single-end reads of 75bp. Reads were aligned to the human reference genome GRCh37 using STAR v2.4.2a46. Picard’s AddOrReplaceReadGroups v1.98 (http://broadinstitute. github.io/picard/) was used to add read groups to the BAM files, which were sorted with Sambamba v0.4.547 and transcript abundances were quantified with HTSeq-count v0.6.1p148 using the union mode. Subsequently, reads per kilobase per million mapped reads (RPKMs) were calculated with edgeR’s RPKM function49. hiPSC-CM RNAseq. Total RNA was isolated from PLN R14del hiPSC-CMs at 72h post-treatment with BiX (0.1 μM) or DMSO control. Sequencing libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Sequencing was carried out on an Illumina HiSeq platform. For each sample 30 to 40 million 150-base paired-end reads were acquired, and data were analyzed using the ENCODE-DCC RNA-seq pipeline (https://github.com/ENCODE-DCC/rna-seq-pipeline). Briefly, reads were aligned to the human genome (GRCh38) using STAR, version 2.5.1b, and gene and transcript quantifications were performed by RSEM (1.2.31). Differential expression analysis was performed in R version 3.4 using the DESeq2 package50. Heat maps of gene expression were generated using the online tool Morpheus.
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