Gs flx

Microbial community RNA was sequenced with Illumina HiSeq2000 (1 × 100 bp; Iowa State University, Ames, IA) in two eight-lane flow cells in accordance with the manufacturer’s instructions. Microbial community DNA was sequenced on a Roche GS-FLX instrument with two FLX plus chemistry (454 Life Sciences) plates, and phage DNA was sequenced on a Roche GS-FLX instrument with one titanium chemistry plate. The microbial metatranscriptome, microbial metagenome, and virome were all used to form a composite assembly, and an assembly of the phage sequences alone was also completed. ORFs in contigs were predicted, sequences were mapped to ORFs and counted (Table S1), and ORFs were annotated by using the FIGfams and SEED subsystems databases (58 (link)), as well as the Resfams (59 (link)) and minCED (60 (link)) tools. The viral sequences were uploaded to VIROME (61 (link)) for annotation. Sequence analysis is described in more detail in Text S1.

Transcriptional profiling of the four BnDGAT1 genes from four developmental stages of the embryo was conducted as described (Troncoso-Ponce et al., 2011 (link)). Briefly, the seeds of greenhouse-grown B. napus were collected at intervals of 18–20, 23–25, 28–30, and 33–35 days after flowering (DAF) and frozen. After collection, total RNA was extracted using TRIZOL (Invitrogen, http://www.invitrogen.com/), followed first by mRNA purification with a commercial kit (Illustra, GE Healthcare, http://www.gehealthcare.com/) then by conversion to cDNA with Superscript III (Life Technologies, http://www.lifetechnologies.com/us/en/home.html) according to the manufacturer’s instructions. For sequence analysis, cDNA was processed with a Roche Library Preparation Kit (http://www.roche.com/) and sequenced with a 454 sequencer (GS FLX, Roche). Expression of the gene family was quantified taking into account the similarity between the gene sequences. For a given gene, the repartitioning of reads is based on the ratio of the number of reads mapped uniquely on this gene to the total number of reads mapped uniquely on the genes sharing the common reads. FPKM = number of reads × (1000/gene length) × (1 million/total number of reads).

A fragment of the 16S rRNA gene encompassing the V3 and V4 variable regions [29 (link)] was amplified with primers 357f (5′ CCT ACG GGA GGC AGC AG 3′, [30 ]) and 786r (5′ AC CAG GGT ATC TAA WCC 3′, this work) with overhangs required for pyrosequencing. The forward primer was used in three versions differing in the multiplex identifier (MID) sequence between the A adaptor and the actual primer. The reaction mix contained the following: 1 ng of template DNA, 10 pmol primers, 0.2 mM dNTP, 1.5 mM MgCl₂, 0.2 U of Taq polymerase, and the appropriate 1× buffer (Fermentas, Lithuania). The cycling conditions were as follows: 95 ºC for 5 min, 30 cycles of: 95 ºC for 30 s, 52 ºC for 30 s, 72 ºC for 30 s; and finally 72 ºC for 2 min. The reactions were carried out in MasterCycler thermocycler (Eppendorf, Germany). Eight independent reactions were performed for each sample.
PCR products were purified with the DNA CleanUp kit (A&A Biotechnology, Poland), pooled in equimolar amounts, vacuum dried, and either sent for sequencing at Max Planck Molecular Genetics Institute sequencing service (Berlin, Germany—Olkusz samples) or sequenced at the Chair of Genetics, University of Silesia (Katowice, Poland—Alwernia ones). Pyrosequencing was performed with the use of Titanium chemistry on GS FLX and GS Junior instruments (Roche, USA) as described in the manufacturer’s protocols.