454 gs flx platform

Four tissues of C. oleifera, including tender shoots, young leaves, flower buds and flowers, were harvested in 2010 from East Park of Kunming Botanic Garden, Yunnan Province, China. All necessary permits were obtained from Wei-bang Sun, who is the director of the Kunming Botanical Garden, the Chinese Academy of Sciences. All samples were flash frozen in liquid nitrogen and stored at −80°C for RNA extraction. Total RNA was extracted by a modified CTAB method. The quality and quantity of total RNA were analyzed using agarose gel electrophoresis and Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies, CA). cDNA library construction and normalization were performed as described previously [44] (link). All libraries were combined into a single pool and sequenced using the 454 GS-FLX platform (Roche, IN, USA).

The taxonomic diversity present in the samples was assessed by high-throughput sequencing of partial 16S rDNA gene amplicons on a Roche 454 GS FLX platform. For this, the DNA extracted from each sample was quantified and amplified with barcoded universal primers for the V4 and V5 regions of the 16S rRNA gene as described previously [29] (link). The Qiime pipeline version 1.5.0 [30] (link) was used to process and analyse the 16S rRNA sequence data. Sequences were binned by samples using the sample-specific barcode sequences, trimmed of the barcode and primer sequences, filtered (sequences required a length ≥300 bp, no undetermined bases, and a perfect match to the barcode and PCR primer), and denoised. Sequences were clustered into operational taxanomic units (OTUs) using UCLUST [31] (link) with a 97% sequence identity threshold. Chimeric sequences were identified with ChimeraSlayer [32] (link) and excluded from further analysis. OTUs were assigned taxonomy using the Ribosomal Database Project (RDP) classifier (minimum confidence of 80%) [33] (link) and the Greengenes database [34] (link). Based on the number of sequences obtained per sample (see results), the relative OTU abundance for each sample was determined at an even depth of 11070 sequences per sample (randomly picked without replacement; OTUs observed less than five times were excluded from this analysis).

The V3-V5 regions in the 16S rRNA gene of Escherichia coli were amplified by PCR from extracted DNA using sample-unique DNA bar-coded primers (forward: 5′-CCGTCAA- TTCMTTTGAGTTT-3′, reverse: 5′-ACTCCTACGGGAGGCAGCAG-3′) [43 (link)]. The PCR reaction program was as follows: 3 min initial denaturation at 94°C, 20 cycles of denaturation at 94°C for 1min, annealing from 65°C to 55°C (1°C reduction for every two cycles) for 1 min, elongation at 72°C for 1 min, and the final extension at 72°C for 6 min [44 (link)]. Then, the resulting products were verified via gel electrophoresis. DNA pyrosequencing was carried out on 454 GS FLX+ platform (Roche, Branford, CT, USA).

The fungal internal transcribed spacer (ITS, ITS1-5.8S-ITS2) of nuclear ribosomal DNA sequences was amplified by using a set of primers designed by adding a 10-nucleotide barcode to ITS1F and ITS4 primer sets [34 ]. The 20 μl reaction mixture contained 10 ng of template DNA, 4 μl of 5×buffer, 2 μl of 2.5 nM dNTP, 0.8 μl of Fastpfu Polymerase, 2 μM of each primer and ddH₂O. The PCR amplification consisted of an initial denaturation at 95°C for 2 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 5 min. The PCR products were purified with an AxyPrepDNA Gel Extraction Kit (Axyen Scientific, Inc.) according to the manufacturer’s instructions. The purified PCR amplicons from each sample were mixed and then pyrosequenced by using the 454 GS FLX + Platform (Roche Applied Science, USA) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China. The raw sequence reads were deposited in the NCBI sequencing read archive (SRA) under accession no. SRP049024.

The V1–V3 region of the 16S rDNA gene was amplified with barcoded primers (8F/533R) (Wu et al., 2012 (link)). Replicated PCR products of each sample were pooled and separated by 2% agarose gel electrophoresis, then purified with a DNA gel extraction kit (Axygen, China). Prior to sequencing, the concentrations of purified amplicon DNA were determined using a QuantiFluorTM-ST Fluorometer (Promega, Madison, WI, United States). Equal amounts of the PCR products were mixed together and sequenced on a 454 GS FLX + platform (Roche Applied Science, Indianapolis, IN, United States) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

Testes were dissected from 2,000 early (up to 4-h old) pupae and from 200 third instar and early fourth instar larvae, and stored in RNAlater (Ambion) at −20 °C until RNA isolation. RNA was isolated using RNeasy Mini kit (Qiagen), followed by poly-A+ mRNA isolation using Oligotex mRNA Mini kit (Qiagen). The mRNA was treated with TURBO DNase (Ambion) and its integrity evaluated using the Agilent 2100 Bioanalyzer. Then, the mRNA was used to synthesize double-stranded cDNA using the SMART™ PCR cDNA synthesis kit (Clontech) according to the manufacturer’s recommendation. Fragmentation of 1 μg of double-stranded cDNA by nebulization, fragment ends polishing, adaptor ligation, isolation and estimation of the single-stranded template library concentration, emulsion PCR with library beads, loading on the 454 plate, and pyrosequencing on the 454 GS FLX platform (Roche 454 Life Sciences) using Titanium chemistry was performed according to the published protocol (Margulies et al. 2005 (link)). Sequence reads were mapped to the AgamP4.1 gene models using GS Reference Mapper v2.3 (Roche). Raw read counts per gene were normalized and converted into RPKM expression values as described above, and are presented in supplementary table S5, Supplementary Material online.

The normalised library prepared by Evrogen was sent to the Centre for Genomic Research (University of Liverpool, UK) for sequencing using 454 GS FLX platform (Roche, Basel, Switzerland). Data were quality checked using FastQC, and quality trimmed using clean_reads (http://bioinf.comav.upv.es/clean_reads) and all default settings. All bacterial reads were removed by aligning against all bacterial genomes downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). Finally the SMART vector was removed using fastx_clipper (http://hannonlab.cshl.edu/fastx_toolkit/index.html) all sequences uploaded to SRA as study SRP028239.