Human induced pluripotent stem cells-derived cerebellar organoid for modelling Friedreichs ataxia disease
RNA was extracted (three samples for each group, each sample consisting of three organoids) using the RNeasy Mini Kit (Qiagen). RNA was quantified using the Agilent RNA ScreenTape System (Agilent) on a 4200 TapeStation (Agilent). RNA (500ng, RIN >8) was used to prepare RNA-seq libraries with the KAPA mRNA HyperPrep Kit (Roche) according to the manufacturers protocol. Sequencing libraries were quantified by qPCR using the KAPA library quantification kit (Roche) using QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). All samples were normalized according to concentration and pooled. Libraries were loaded and sequenced using the Illumina Novaseq 6000 system. Bioinformatics analysis for bulk RNA-seq was carried out using the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov) using R language 3.6.0 (https://cran.r-project.org/). Bulk RNA-Seq samples were quality trimmed using Trimmomatic 0.36 and the TruSeq3 paired-end adapters. STAR aligner 2.7.6a followed by HTSeq-count 0.9.1 produced deduplicated counts of reads in genes. UCSC gene counts were normalized using the default median-of-ratios method in DESeq2 1.24.0. Differential expression tests used the lfcShrink function and gene set enrichment was performed using Enrichr API package enrichR 1.0.