Gene expression profiling using RNA sequencing

Total RNA extracted from tissue samples obtained from patients 7 and 8 (Table I) were treated with DNase to remove genomic DNA. The NEB Next rRNA Depletion kit (human/mouse/rat) (New England Biolabs Inc.; cat no. E6310S) was subsequently used to remove the ribosomal RNA from the samples. The mRNA was converted into cDNA using the Ultra RNA Library Prep kit (New England Biolabs Inc.; cat no. 7530L) and subsequently sequenced using the Illumina MiSeq system. The CLC Genomics Workbench version 11 software (www.qiagenbioinformatics.com/products/clc-genomics-workbench) was used for data normalization and differential gene analysis, with HG19 serving as the reference genome.