title: Transcriptome study in Sicilian patients with autism spectrum disease
design: Transcriptome study in Sicilian patients with autism spectrum disease
library_construction_protocol: This case-control study consists of a mRNA analysis on peripheral blood mononuclear cells (PBMCs) performed by RNA-seq in 25 participants of Sicilian ancestry: 12  patients and 13 age- and sex-matched healthy controls, all recruited at the Oasi Research InstituteIRCCS of Troina (Italy). Total RNA was extracted using Zymo Research, according to the manufacturer's instructions. The RNA was transcribed to cDNA using oligo (dT) primers and the GeneAmp RNA PCR Core Kit (Applied Biosystems) and the cDNA was amplified via real-time PCR using Power- SYBR Green PCR Master Mix (Promega) on the 7500 Fast Real-Time PCR System (Applied Biosystems). RNA concentration and purity were evaluated using NanoDrop™ 2000/2000c (Thermo Fisher Scientific, Waltham, MA, USA), whereas sample integrity was analyzed using Tape Station 4200 (Agilent Technologies, Santa Clara, CA, USA) using an RNA Screen Tape Assay. Indexed libraries were prepared from 1 µg/ea purified RNA using the TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) Library Prep Kit according to the manufacturer's instructions. After the enrichment of mRNA using oligo dT magnetic beads and fragmentation, cDNA synthesis was performed, followed by adapter ligation and PCR amplification. For library quantifications, TapeStation 4200 (Agilent Technologies) was used. Indexed libraries were pooled in equimolar amounts, with a final concentration of 1.5 nM.