small RNA-seq of Juglans californica
Individual indexed libraries were prepared from 1 g of purified RNA using the TruSeq SmallRNA Sample Prep Kit (Illumina, USA) according to the manufacturers instructions. Both RNA samples and final libraries were quantified by using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer RNA Nano assay (Agilent technologies, Santa Clara, CA). Libraries were then processed with Illumina cBot for cluster generation on the flow cell, following the manufacturers instructions and sequenced on single-end mode 50 bp inmultiplexingon HiSeq2500 (Illumina, San Diego, CA). The CASAVA 1.8.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. Raw data sequence underwent quality controls analysis by using FastQC release 0.11.5 version [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/] and MultiQC V 0.9 49. The Illumina small-RNA adapter was clipped using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit) and sequences shorter than 15nt after trimming were removed and the remaining reads were trimmed on the 3p and 5p ends in order to remove low-quality bases. At the end of this procedure, BLASTn v 2.2.30 with parameters -task megablast, -perc_identity 100 was used to compare the remaining reads with the Rfam database (Rfam 11.0) 50. This stringent step removed non-coding RNA (rRNA, tRNA, snRNA, snoRNA) and degraded fragments of mRNA.