title: Ion Torrent Proton sequencing; Wide-ranging analysis of microRNA profiles in sporadic amyotrophic lateral sclerosis using next-generation sequencing
design: Wide-ranging analysis of microRNA profiles in sporadic amyotrophic lateral sclerosis using next-generation sequencing
library_construction_protocol: Peripheral blood and neuromuscular junction from a total of 70 patients with sporadic amyotrophic lateral sclerosis (n = 45), and healthy controls (n = 25) was obtained (see Table 1 Supplementary file). Blood samples from healthy controls and from patients with ALS were obtained from the Monaldi Hospital, Naples, Italy and neuromuscular junction from ALS patients were purchased from Edinburgh Tissue Bio Bank. Detailed patient characteristics are listed in Table 1 Supplementary file. ALS patients have been diagnosed as clinically definite sALS with the support of electroneuromyography (ENMG). The onset of the disease was 1–2 years before diagnosis. The mean age of patients and controls was 62 ± 10 years.  RNA extraction was performed as in De Felice et al (2014). Briefly, we obtained ten micrograms of total RNA from peripheral blood samples drawn from patients and controls in the morning. Trizol (Invitrogen, no. 15596-026) method has been used for isolation and purification of RNA from whole blood and from neuromuscular junction samples. RNA was isolated including a DNase digestion step. These standardized RNA isolation procedures guarantee high-quality RNA. Using the Agilent 2100 Bioanalyzer platform (Agilent Technologies) RNA samples were quality-checked documenting the identification of 18-S and 28-S ribosomal RNA (rRNA) peaks .The yields were 8–15 μg, and the RNA Integrity Number (RIN) was between 7.2 and 10. Such results fulfilled published data, reporting that RNA with a RIN >6 is of sufficient quality for miRNA microarray experiments as well as gene and microRNA expression-profiling experiments   We performed the enrichment of small RNA fraction from total RNA utilizing the Magnetic Bead Cleanup Module (Life Technologies, Carlsbad, CA, USA). Using Ion Total RNA-Seq kit v2 Life Technologies, Carlsbad, CA, USA, 50 ng of enriched small RNA sample was ligated to proper adapters and reverse transcribed to cDNA.  cDNA samples were size-selected from 50 to 300 nt  (Magnetic Bead Cleanup Module, Life Technologies), and PCR amplified using 5' primers containing a unique index barcode (Ion XpressTM RNA-Seq Barcode 1–16 Kit, Life Technologies, Carlsbad, CA, USA). We assessed the yield and size distribution of the small RNA libraries using the Agilent 2100 Bioanalyzer TM instrument and the Agilent HS DNA kit. We amplified similarly pooled libraries on Ion SphereTM Particles (ISPs) provided by the Life Technologies Ion PI Hi-Q Chef kit. All the steps to perform the emulsion PCR and the loading of the pooled libraries in the Life Technologies ION PI Chip v3 were performed automatically on Life Technologies ION Chef Instrument. Eight libraries were pooled in one chip.
attributes.tag: Experimental Factor: disease
attributes.value: sporadic amyotrophic lateral sclerosis
attributes.tag: Experimental Factor: organism part
attributes.value: muscle tissue
attributes.tag: Experimental Factor: sampling site
attributes.value: neuromuscular junction