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Produced Water Exposure Alters Bacterial Response to Biocides Amit Vikram, Daniel Lipus, and Kyle Bibby Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5036915 • Publication Date (Web): 03 Oct 2014 Downloaded from http://pubs.acs.org on October 7, 2014
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Environmental Science & Technology
Produced Water Exposure Glutaraldehyde Tolerance
Hypochlorite tolerance
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Produced Water Exposure Alters Bacterial Response to Biocides Amit Vikram1,2, Daniel Lipus1,2, and Kyle Bibby1,2,3* 1
National Energy Technology Laboratory, Pittsburgh, PA 15236, 2Department of Civil and Environmental Engineering, and 3Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA *
Corresponding author
*
Corresponding Author: Kyle Bibby, 709 Benedum Hall, Pittsburgh, PA 15261
[email protected], 412-624-9207 Keywords. RNA-seq, glutaraldehyde, hypochlorite, biocide tolerance, flowback water, produced water, hydraulic fracturing, microbiology, Pseudomonas, Marinobacter
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ABSTRACT Microbial activity during the holding and reuse of wastewater from hydraulic fracturing
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operations, termed produced water, may lead to issues with corrosion, sulfide release, and
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fouling. Biocides are applied to control biological activity, often with limited efficacy,
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which is typically attributed to chemical interactions with the produced water. However,
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it is unknown whether there is a biologically driven mechanism to biocide tolerance in
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produced water. Here, we demonstrate that produced water exposure results in an
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enhanced tolerance against the typically used biocide glutaraldehyde and increased
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susceptibility to the oxidative biocide hypochlorite in a native and a model bacteria and
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that this altered resistance is due to the salinity of the produced water. In addition, we
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elucidate the genetic response of the model organism Pseudomonas fluorescens to
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produced water exposure to provide a mechanistic interpretation of the altered biocide
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resistance. The RNA-seq data demonstrated the induction of genes involved in osmotic
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stress, energy production and conversion, membrane integrity, and protein transport
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following produced water exposure, which facilitates bacterial survival and alters biocide
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tolerance. Efforts to fundamentally understand biocide resistance mechanisms, which
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enable the optimization of biocide application, hold significant implications for greening
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of the fracturing process through encouraging produced water recycling. Specifically,
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these results suggest the necessity of optimizing biocide application at the level of
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individual shale plays, rather than historical experience, based upon produced water
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characteristics and salinity.
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INTRODUCTION
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High volume hydraulic fracturing (‘fracking’) coupled with horizontal drilling has
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emerged as a significant enabling technology for hydrocarbon extraction from
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unconventional reservoirs. In high-volume hydraulic fracturing operations, large volumes
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(15-20 million liters 1) of fracture fluid are injected under high pressure to fracture the
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shale formation and increase formation permeability and gas flow rates 2. Fracture fluid is
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typically composed of approximately 90% water and 9% sand used to support the created
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microfractures. The remaining 1% is made up of chemicals to assist in the fracturing
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process, such as viscosity modifiers, friction reducers, anti-scalants, and biocides 2.
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Following the fracture process, a portion of the fracture fluid (5-100%) mixed with
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subsurface brine returns to the surface as produced water 2, 3. Characteristics of produced
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water vary by geologic formation; however, produced water is generally saline to hyper-
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saline (20,000 to greater than 250,000 mg/l total dissolved solids) with elevated
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concentrations of divalent cations 1. Management and disposal of produced water is of the
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utmost operational and environmental concern. Recently, reuse of produced water in
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future hydraulic fracturing operations has emerged as a desirable management approach
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to reduce environmental and logistical concerns associated with produced water disposal
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and sourcing of freshwater for fracturing fluid. In the near future, reuse of produced water
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may become more prevalent as deep well injection, a commonly used approach for
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produced water disposal, was recently linked to increased local seismic activity 4.
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Microbial activity during the holding and reuse of produced water is highly
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undesirable, and can lead to the corrosion of piping and equipment or fouling (sulfide
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release) 2. Biocides are typically used to control microbial growth in fracturing fluid and
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produced water. The biocides glutaraldehyde, sodium hypochlorite (NaOCl) or amine
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type compounds (e.g. cocodiamine) are often used alone or in combination to control
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biological activity
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populations that were able to survive biocide treatment in fracturing fluid and produced
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water 1, 6-9. These reports have raised concerns about the biocide resistance and tolerance
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of microbes present in produced water. Additionally, there are industrial concerns that
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reuse of produced water in future hydraulic fracturing operations can potentially lead to
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microbial growth and clogging of shale fractures 8, 10-12.
2, 5
. However, anecdotal reports and studies have reported bacterial
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Despite the substantial investment in control of biological activity in wastewater
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from hydraulic fracturing operations, the mechanisms microorganisms use to survive in
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saline produced water and possible biological mechanisms of biocide tolerance are not
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well understood
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microbial populations in produced water
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survival mechanisms in saline produced water will allow the development and
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optimization of treatment and disinfection methods for produced water, limiting
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undesirable microbial activity and encouraging produced water reuse.
5, 7, 13
, hindering the optimization of biocide application to control 5, 14, 15
. Understanding microbial growth and
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The goal of the current study was to demonstrate a biological, rather than
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physiochemical mechanism for biocide inefficacy in produced water from hydraulic
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fracturing. To complete this goal, we investigated the change in biocide efficacy and gene
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expression in response to produced water exposure in the model organism Pseudomonas
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fluorescens. P. fluorescens was chosen as a model organism for these studies as it is a
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model environmental Pseudomonas, which are common and abundant inhabitants of
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produced water, with a sequenced and well-characterized genome. Previous 16S rDNA
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sequence explorations from the Barnett, Antrim and Marcellus shales have shown that
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each formation has the Pseudomonas genus in relatively high abundance within its
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unique characteristic microbial diversity 6, 7, 13, 1. Pseudomonas spp. have previously been
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shown to cause biofouling and corrosion, operational concerns in produced water
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management
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exposure, we exposed P. fluorescens to produced water and examined biocide tolerance
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and gene expression via RNA-seq. Biocide tolerance results were further confirmed using
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a native produced water isolate Marinobacter sp. Biocide tolerance was also tested in an
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additional produced water and laboratory made saline water for both bacteria.
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MATERIALS AND METHODS
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Bacterial strain and culture conditions
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Pseudomonas fluorescens Migula (ATCC 13525) was purchased from ATCC, maintained
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on cetrimide agar plates, and cultured in Luria Bertani broth (Thermo Fisher Scientific,
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Pittsburgh, PA) at 25°C and 200 rpm agitation. The Marinobacter species was isolated
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from produced water as detailed in the supplementary methods and maintained on high
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salt tryptic soy agar (high salt TSA) plates. High salt TSA plates were prepared by adding
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810 mM NaCl, 68.5 mM CaCl2 and 7 mM MgCl2 to standard TSA.
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Produced water samples were obtained from Marcellus Shale natural gas wells in
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southwestern Pennsylvania, and their chemical parameters are shown in Table 1.
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Produced water sample 1 (PW1) was sampled from an actively flowing well, filter
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sterilized using a 0.2 µm membrane filter, and stored in the lab in the dark until use.
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Produced water sample 2 (PW2) was collected from a produced water holding pond, filter
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sterilized, stored in the lab in the dark until use.
16, 17
. To investigate altered biocide tolerance due to produced water
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Survival, Biofilm Formation and Biocide Susceptibility Assay
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Survival and biofilm formation assays were conducted as described in supplementary
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methods. The minimum inhibitory dose (MIC) of glutaraldehyde for P. fluorescens was
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determined by broth dilution and found to be approximately 70 mg/L in produced water.
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Similarly, MIC for NaOCl was found to be 10 mg/L (free chlorine equivalent).
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Subinhibitory doses of 60 mg/L glutaraldehyde and 8 mg/L NaOCl were chosen for the
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study. Three overnight cultures of P. fluorescens and Marinobacter sp. were grown in LB
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broth or LB broth supplemented with 810 mM NaCl, 68.5 mM CaCl2 and 7 mM MgCl2.
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The cells were pelleted by centrifugation at 1200 x g and approximately 106 cfu/ml of
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cells were resuspended in PBS (Hyclone laboratories, Logan, UT; see table S1 for
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composition) as a control and either PW1, PW2, or laboratory derived saline water (810
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mM NaCl, 68.5 mM CaCl2 and 7 mM MgCl2 in deionized water) and incubated for 1
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hour at room temperature without shaking. All samples and controls were filter sterilized.
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The cells were collected post-incubation by centrifugation at 1200 x g for 5 minutes and
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resuspended in PBS containing 60 mg/L glutaraldehyde or 8 mg/L NaOCl (free chlorine
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equivalent) for 1 hour. Serially diluted aliquots (100 µl) were plated on cetrimide agar or
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high salt TSA plates and colonies were counted after 24 hours. Tests for all conditions
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were conducted in biological triplicate from individual seed cultures. Controls (0 ppm
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biocide), were run for all conditions and did not demonstrate a statistical difference by
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treatment.
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Produced Water Exposure for Transcriptome Analysis
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Two sequencing runs of three pooled biological replicates (for a total of 6 biological
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replicates) each were performed and analyzed individually to obtain the final analysis.
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For each sequencing run, three overnight broth cultures of P. fluorescens were diluted
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100 fold in fresh LB broth and incubated at 25°C with agitation at 200 rpm and grown to
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an OD600 of approximately 0.3 (six generations, 10 mm path length measured on a
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Genesys 10S spectrophotometer, Fisher Scientific, Pittsburgh, PA). The cells were
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collected from 10 ml cultures by centrifugation at 2000 x g and resuspended in 1 ml PW1
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or PBS and incubated at room temperature for 1 hour. Following exposure, the cells were
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pelleted at 2000 x g, 4 °C for 5 minutes. RNA was immediately extracted with TRIZOL
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(Life Technologies, Carlsbad, CA) and treated with Turbo DNase (Life Technologies,
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Carlsbad, CA). The RNA quality was checked on a 1.3% agarose gel by inspecting the
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16S and 23S rRNA bands, quantified using Qubit (Life Technologies, Carlsbad, CA) and
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stored at -80 °C until sequencing.
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For each replicated sequencing run, RNA samples from three biological replicates
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of PW1 or PBS were pooled and processed using the ScriptSeq™ Complete Kit
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(Bacteria) (Epicenter Biotechnologies, Madison, WI) according to the manufacturer’s
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instructions. Briefly, 1 microgram of DNase treated RNA samples was treated with
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RiboZero to remove ribosomal RNA, purified by ethanol precipitation and dissolved in
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10 µl RNase free water. rRNA depleted samples were mixed with RNA fragmentation
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solution and cDNA synthesis primer provided in the ScriptSeq kit and cDNA was
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synthesized using StarScript Reverse Transcriptase at 25°C for 5 minutes followed by 42
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°C for 20 minutes. The cDNA was di-tagged using Terminal Tagging Premix and
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purified using Agencourt AMPure Purification kit (Beckman Coulter, Indianapolis IN).
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Finally, the di-tagged cDNAs were PCR amplified using Failsafe PCR enzyme and
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barcoded reverse primers provided with the ScriptSeq kit as per manufacturer’s protocol.
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The amplified ditagged cDNAs were purified using Agencourt AMPure XP beads and
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quantified using Qubit.
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Sequencing
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The di-tagged, barcoded cDNA samples were sequenced on an Illumina MiSeq sequencer
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(San Diego, CA). Equimolar amounts of cDNA from PBS and PW1 treated samples were
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pooled and diluted to 2nM. Each pooled sample was then denatured using fresh 0.2 N
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NaOH for 5 minutes at room temperature and further diluted according to manufacturer’s
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instructions. The final library was spiked with 5% PhiX genome control and sequenced
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using a MiSeq V2 reagent kit (300 cycles) (Illumina, San Diego, CA).
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Bioinformatics
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Differential gene expression analysis was conducted using RNA-seq algorithm
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the CLC Genomics Workbench 6.5.1 (CLC Bio, Aarhus, Denmark). Reads with quality
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scores
