Sesión para el desarrollo de las estrategias de compensación

Genomic RNA and DNA was extracted from Butyrivibrio co-cultures using a

modified version of a liquid N2 and grinding method from Lueders et al., (2004).

RNA handling practises

To prevent RNase contamination during RNA experiments, equipment, surfaces and pipettes were wiped with Ambion RNaseZap (Thermo Fisher Scientific Inc., Waltham, MA, USA) and only RNase-free pipette tips and plastic-ware were used. All

solutions and buffers were made using RNAse-free, DEPC-treated H2O and all equipment

for RNA extractions were made RNAse-free by heating at 180 ºC for 2 h and autoclaved at 121 ºC for 20 min.

Modified phenol-chloroform nucleic acid isolation with bead beating

The following protocol was for 10 g of frozen culture sample harvested as described below. The frozen sample was weighed into a 50 mL Falcon tube containing 10 mL of RNA Bacterial Protect Reagent (Qiagen, Hilden, Germany), and left to thaw on ice for 2

h. The thawed sample was centrifuged at 15,000 × g for 10 min at 4 ºC and the supernatant

discarded. The cell pellet was resuspended in 1 mL of Extraction buffer A (200 mM NaCl,

20 mM EDTA), 420 μL of 20% (w/v) SDS and 1 mL of a mixture of phenol: chloroform:

isoamyl alcohol solution. The resuspended mixture was transferred to 2 mL screw cap vials (Sarstedt, Nümbrecht, Germany) containing 500 mg of 0.1 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK, USA), and cells were disrupted by bead-

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beating samples twice using a Mini-Beadbeater-96 (BioSpec, Bartlesville, OK, USA) for 4 min at full speed. Samples were stored at 4 ºC overnight followed by three rounds of

bead-beating, centrifuged at 10,000 × g for 5 min and the top layer was transferred to a

fresh 1.5 mL Eppendorf tube. An equal volume of isopropanol and 0.1 volume of 3 M sodium acetate (pH 5.5) were added, gently mixed and stored at -20 ºC overnight. The

RNA/DNA was harvested by centrifugation at 10,000 × g for 30 min at 4 ºC, the

supernatant was discarded and residual isopropanol was carefully removed using a

pipette. The RNA/DNA pellets were washed twice with 500 μL ice-cold 70% (v/v)

ethanol and centrifuged at 10,000 × g for 10 min at 4 ºC after each wash (Lueders et al.,

2004). The washed pellets were air-dried for 20 min and re-suspended in 100 μL of

nuclease-free water (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RNA/DNA samples were stored at -85 °C and used for qPCR analyses.

DNAse treatment

Following RNA/DNA extraction, the residual double and single stranded DNAs were removed from the RNA samples using the Baseline-ZERO DNase (Epicentre

Technologies, Madison, WI, USA) treatment kit according to the manufacturer’s

instructions. To concentrate the purified RNA, samples were precipitated by addition of 1:10 volumes of 5 M ammonium acetate and 2.5 volume of 100% ethanol, mixed and

incubated at -20 ºC for 30 min. Samples were centrifuged at 16,000 × g for 15 min at 4

ºC and the supernatant discarded. The pellets were washed with 500 μL of ice-cold 70%

(v/v) ethanol and centrifuged again as before after each wash. The washed pellets were

air-dried for 20 min and re-suspended in 100 μL of nuclease free water (Thermo Fisher

Scientific Inc., Waltham, MA, USA). RNA was stored at -85 °C until required. DNA

elimination was verified by a lack of product from PCR amplifications of the 16S rRNA

gene.

RNA purification and quantification

Following DNAse treatment, the MEGAClear kit (Thermo Fisher Scientific Inc.,

Waltham, MA, USA) was used to purify mRNA following the manufacturer’s

instructions, which also removes tRNAs and 5S rRNA. In addition, the elution procedure

was repeated with a second pre-heated 50 μL aliquot of Elution solution. RNA yield and

quality were measured using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara,

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RNA integrity was also determined using agarose gel electrophoresis under RNase free

conditions to detect distinct 16S and 23S ribosomal RNA (rRNA) bands. RNA samples

were stored at -85 ºC prior to sending for RNA sequencing at the Beijing Genomics Institute (BGI, China).

cDNA library preparation for RNA-seq

cDNA was synthesised from the DNase-treated RNA samples at the BGI (Beijing, China) and an overview of the experimental pipeline for transcriptome preparation is outlined in Figure 2.1. Briefly, pre-treatment of the RNA sample involved thawing samples on ice, centrifugation and mixing prior to quality testing. Concentration of RNA

was determined using NanoDrop® _{ND-1000 for OD}

260/280nm and OD260/230nm and

electrophoresis profile analysis in the the RNA 6000 Nano Chip Kit for capillary electrophoresis (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA, USA), with

the quality score determined by RIN analysis of for 23S/16S rRNA peaks. After total

RNA concentration and quality was determined, the remaining cDNA synthesis and sequencing were done by BGI. Firstly, fragmentation buffer was added for disrupting mRNA to short fragments. These fragments and random hexamer-primers were then used to synthesise the first cDNA strand. The second cDNA strand was synthesised using buffer, dATPs, dGTPs, dCTPs, dUTPs, RNase H and DNA polymerase I, respectively, after removing dNTPs (including dTTP). Short fragments (100 to 500 nucleotides) were

purified using QIAquick® _{PCR purification kit (Qiagen, Hilden, Germany) and dissolved}

with Elution Buffer (EB) prior to addition of poly(A). Fragments were ligated to sequencing adapters and the Uracil N-Glycosylase (UNG) enzyme was used to degrade the second strand cDNA (containing dUTP). The product was purified using the MiniElute PCR purification kit (Qiagen, Hilden, Germany) prior to PCR amplification. The generated libraries were then sequenced using Illumina HiSeq 2000 technology (BGI, Beijing, China).