Supplementary Material and Methods
Yergeau et al. “Microbial expression profiles in the rhizosphere of willows depend on soil contamination”
Ion Torrent 16S rRNA sequencing
Reverse-transcriptase-PCR of the partial 16S rRNA was performed using the universal primers F343 (5’TACGGRAGGCAGCAG-3’) and R533 (5’- ATTACCGCGGCTGCTGGC-3’) containing the 10-bp multiplex
identifiers (MID) and adaptor sequences for Ion Torrent sequencing described previously (Yergeau et al.,
2012a; Bell et al., 2013). Reactions were performed using a Qiagen OneStep RT-PCR kit (QiaGen,
Valencia, CA) in 25-μl volumes containing 2 μl of template RNA (50-100 ng/µl), 5µl of 5X reaction buffer,
0.6 μM of each primer, 0.4 mM of dNTPs and 1 μl of enzyme mix. Cycling conditions involved a reversetranscriptase step at 50°C for 30 min, an initial 15 min denaturing step at 95°C, followed by 15 cycles of
30 s at 94°C, 30 s at 55°C, and 60 s at 72°C, and a final elongation step of 10 min at 72°C. All PCR
products were purified on agarose gels using the QIAquick Gel Extraction Kit (Qiagen) and quantified
using the PicoGreen dsDNA quantitation assay (Invitrogen, Carlsbad, CA). All amplification reactions
from the 24 different samples were pooled in an equimolar ratio and sequenced together. A total of
3.50x107 molecules were used in an emulsion PCR using the Ion OneTouch 200 Template Kit v2 (Life
Technologies) and the OneTouch instruments (Life Technologies) according to the manufacturer’s
protocol. The sequencing of the pooled library was done using the Ion Torrent Personal Genome
Machine (PGM) system and a 316 chip with the Ion Sequencing 200 kit according to the manufacturer’s
protocol. Sequences were binned by MID, after which MIDs were trimmed from each sequence.
Sequences were trimmed when the average quality over a 5 bp window dropped below a Phred score of
20 or at the first occurrence of an “N” or of a >8bp homopolymer. Sequences shorter than 100 bp, were
then filtered out of the dataset. Taxonomic identities were assigned to sequences using the
“multiclassifier”, which is the local multi-sample version of the RDP Pipeline Classifier
(http://pyro.cme.msu.edu/). Weighted-normalized Unifrac distances between each sample pair were
calculated using the FastUnifrac website (Hamady et al., 2010) based on the GreenGene core dataset.
For OTU calculations, sequence data were randomly normalized to 10,000 sequences and were then denoised using the procedure of Quince et al. (2011). The raw sequence data have been submitted to the
NCBI SRA database under accession No. SRP028579 (NCBI BioProject PRJNA213915).
illumina mRNA sequencing
rRNA was subtracted following the protocol described by Stewart et al. (2010). After subtraction, a 227
bp control RNA transcribed from the pSPT18 vector (positions 2867-3104 and 1-70) was added in a
1:2000 ratio (on a nanogram basis) to the total rRNA-subtracted RNA. This mixture was then reversetranscribed using the SuperScript III kit (Invitrogen). illumina libraries were prepared following the
protocol of Meyer and Kircher (2010), with indices 1 to 24 pooled together and sent for eight lanes of
Illumina HiSeq 2000 paired-end 2x101 bp sequencing at McGill University and Génome Québec
Innovation Center, Montréal, Canada. Resulting data were split into 384 files (24 samples x 2 reads x 8
lanes). The raw sequence data have been submitted to the NCBI SRA database under accession No.
SRP028579 (NCBI BioProject PRJNA213915). Data from the different lanes were pooled together and the
resulting 48 files were filtered in pairs using a custom-made Perl script. Sequences were trimmed at the
first occurrence of a low quality base (below phred-like score of 20) or when an adapter sequence was
found. Sequences shorter than 50 bp were filtered out and if one of the sequences of the pair was
rejected, then the second one was also automatically rejected. The resulting high-quality sequences
were submitted to MG-RAST 3.0 (Meyer et al., 2008) for automated annotation. Paired-end files were
combined and overlapping reads were assembled within MG-RAST. Filtered and annotated sequence
data are available through MG-RAST accessions 4512569-4512592. The number of sequences related to
the pSPT18 vector in the filtered datasets was obtained by Blast using an e-value cutoff of 10-25 and this
number was used to normalize the number of transcripts using the method of Moran et al. (2013).
Quantitative reverse-transcriptase real-time PCR (RT-qPCR)
RT-qPCR was performed in 20 µl volumes using the iScript One-Step RT-PCR kit with Sybr green (Bio-Rad
Laboratories, Hercules, CA) on a Rotor-Gene 3000 apparatus (Corbett Life Science, Sydney, Australia), as
previously described (Yergeau et al., 2009; 2010; 2012b). Reactions were set-up as per the
manufacturer’s instructions, with 0.01 ng of total soil RNA and primers Eub338/Eub518 (Fierer et al.,
2005). The amplification procedure was as follows: cDNA synthesis for 10 min at 50°C, reverse
transcriptase inactivation for 5 min at 95°C, PCR cycling and detection (40 cycles) for 10 s at 95°C, 15 s at
53°C and 15 s at 72°C (acquiring signal at the end of this step). Standards were made from 10-fold
dilutions of linearized plasmids containing a full length 16S rRNA gene that was cloned from an isolated
bacterial strain. Several no-reverse-transcriptase and no-template controls were carried out and yielded
no detectable signals.
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