title: Human Gut microbiota, Sample N3, Healthy control
design: We investigated fecal gut microbioma signature of 8 patients affected by Rett syndrome compared to that of 10 age- and sex- matched healthy controls, as well as the correlation with short-chain fatty acids (SCFAs) profile. For gut microbiota, 16s rRNA gene amplicon libraries (targeting V3-V4 hypervariable regions) were performed with a two-step barcoding approach according to Illumina 16S Metagenomic Sequencing Library Preparation. In the first-step PCR, 16S rRNA gene of all bacteria were amplified as described by Klindworth et al.(2013). Then, DNA samples from first PCR step were amplified with dual-index primers using Nextera DNA Library Preparation Kit  Each sample possessed specific barcode sequences at the front and end of the PCR amplicon to discriminate among each other in the pooled library. A pooled library (20 nM) and a PhiX control v3 (20 nM) were mixed with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 12 pM each. The resulting library was loaded on a MiSeq® v2 (500 cycle) Reagent cartridge for obtaining a paired-end 2×250 bp sequencing.