All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting.


INTRODUCTION
Plant sedentary endoparasitic nematodes from Meloidogyne spp. genus (root-knot nematodes), represent a serious threat to the agricultural production (McCarter, 2009). These obligate parasites are attracted by their hosts and after penetration and migration, they establish within the vascular cylinder forming a pseudo-organ, called gall, that include the giant cells (GCs) used for feeding (Escobar et al., 2015). Both molecular and cell biology studies have contributed to a better understanding of the modifications occurring in galls and GCs, where a generalized gene repression takes place in an early-developing stage (Jammes et al., 2005;Barcala et al., 2010;Portillo et al., 2013;Cabrera et al., 2016). However, the mechanisms that contribute to this gene silencing in early developing GCs are still not clear Siddique and Grundler, 2018). The involvement of several microRNA-mediated gene silencing of particular gene targets, such as TCP4, ARF3, TOE1, and MYB33 has been recently reported during the root-knot nematode interaction (Zhao et al., 2015;Cabrera et al., 2016;Medina et al., 2017;Díaz-Manzano et al., 2018). Additionally, the accumulation of rasiRNAs (repeat associated small interfering RNAs; Medina et al., 2018;Ruiz-Ferrer et al., 2018) strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls . However, mechanisms mediated by epigenetic processes such as RNA-directed DNA methylation (RdDM) in this system are still poorly understood. Deep transcriptomic and methylome analysis will be crucial for a detailed information of those putative mechanisms. For this reason, it is essential to obtain RNA and DNA with high integrity and reproducibility among extractions of independent replicates. Moreover, the possibility to simultaneously extract RNA and DNA from the same biological sample constitutes a great advantage for a suitable correlation analysis between transcriptomic and epigenetic changes in the DNA. Furthermore, it minimizes enormously sample collection, which is a tedious and time consuming procedure due to the small size of galls at early infection stages.
Transcriptome analysis, genome sequencing and bisulphite sequencing are examples of a broad list of molecular studies that are performed routinely nowadays. So far, several protocols of simultaneous purification of DNA and RNA have been published on many research fields as, for example, human tissue and blood (Evans et al., 1998;Radpour et al., 2009), cell culture (Vorreiter et al., 2016), fish embryos (Triant and Whitehead, 2009), microbes (Mcllroy et al., 2008;Hill et al., 2015) or infections by viral pathogens in humans (He et al., 2017). However, there is still scarce information about simultaneous extraction of DNA and RNA on the plant field (Xiong et al., 2011;Oliveira et al., 2015;Hazarika and Singh, 2018). Nevertheless, this procedure remains illdefined for the structures and tissues formed during the plant-pathogens interaction, and particularly for the nematodeinduced galls.
Here, we present a protocol that allows a simultaneous extraction and purification of genomic DNA and total RNA from the same plant tissue sample, focused on feeding structures formed by a plant-parasitic nematode, Meloidogyne javanica, in Arabidopsis, and its respective control roots.

PROTOCOL OVERVIEW
In order to obtain galls induced by M. javanica and their respective equivalent control root segments from Arabidopsis, Columbia-0 (Col-0), plants are infected with M. javanica juveniles, samples are hand dissected, collected and quickly frozen in liquid nitrogen. Tissue disruption is performed in a mortar and pestle with a buffer containing 2-mercaptoethanol and homogenized using a QIAshredder R spin column (QIAGEN R ). After the homogenization procedure, the lysate is transferred to an AllPrep R DNA Mini spin column (QIAGEN R ) and centrifuged. We proceed with the RNA purification after a chloroform extraction followed by an incubation with proteinase K. The total RNA is then bound to an RNeasy R Mini spin column (QIAGEN R ), washed and incubated with DNase. It is finally washed several times and eluted. The genomic DNA extraction is then initiated using the remaining AllPrep R DNA Mini spin column (QIAGEN R ). The DNA is incubated with proteinase K and RNase (RNase A, QIAGEN R , Hilden, Germany), washed and eluted. The genomic DNA is finally concentrated using a vacuum concentrator. Quantification and analysis of the RNA and DNA integrity is then performed using a spectrophotometer (NanoPhotometer R Classic, Implen, Munich, Germany), an electropherogram and a gel electrophoresis.

Description of Plant Material and Collection
Nematode populations are maintained in vitro under sterile conditions as described in Díaz-Manzano et al. (2016). Arabidopsis thaliana (L.) Heynh. Col-0 seeds are surface sterilized by soaking in 30% commercial bleach with 0.1% v/v Triton R X-100, for 12 min, sown (at a density of 10 seeds per dish) in 90 mm Petri dishes containing Gamborg B5 medium (Gamborg et al., 1968) supplemented with 15 g L −1 sucrose and 0.6% Daishin Agar (Duchefa Biochemie, Haarlem, Netherlands), pH 7.0, and kept in the dark at 4 • C for 48 h for stratification. Plants are then placed in a growth chamber at 23 • C, 30% relative humidity and a long-day photoperiod (16h/8h; light:dark) where they germinate and grow for 5 days vertically. Roots are then inoculated with 10-15 freshly hatched M. javanica second stage juveniles (J2). Infections are checked every 24 h under a Leica Mz125 (Leica Microsystems, Switzerland) stereomicroscope in order to establish a penetration and infection timeline (described in Portillo et al., 2013). During the first 48 h of infection monitoring, the plates are maintained horizontally in the dark and then placed again vertically and covered with a gauze to avoid excess of light (Olmo et al., 2017). Galls and uninfected root segments are collected as described in Figure 1, according to Portillo et al. (2013). At 3 days post infection (dpi), 300 galls and 1000 control root segments (RCs) were used for each biological independent replicate. For late time points (14 dpi), we collected 250-300 galls and 500 RCs per replicate. This protocol FIGURE 1 | Flowchart of the RNA/DNA extraction procedure. Schematic flowchart for the simultaneous extraction of genomic DNA and total RNA from galls induced by Meloidogyne javanica and its respective control root regments (RC). (A) Plant material is collected, disrupted in a mortar and pestle and homogenized using a QIAshredder R spin column followed by the total RNA purification. The primary root segments of uninfected plants in equivalent positions to that of the galls in infected plants are considered as control root tissue (steps 1-3). (B) RNA purification is followed by purification of the genomic DNA (steps 4-46). Assessment of DNA and RNA purity, quality and integrity is performed in steps 47-49.
focuses on the extraction and purification of DNA and RNA from root tissues and galls in A. thaliana from in vitro cultures. It could potentially be used for the interaction of Arabidopsis with other root pathogens, symbionts or other parasites, yet, specific changes will probably be necessary to adapt it to other plant species or tissues.

Optimization of the Starting Material
Before the nucleic acid's extraction from any plant tissue it is important to optimize the amount of starting plant material which might influence the time, concentrations, volumes, column size and temperature used in all steps. Therefore, it is important to perform an initial experiment to determine the minimum starting material needed. Herein, we have tested different amounts of starting material as well as different tissue disruption methods, TissueLyser II from QIAGEN R or mortar and pestle (data not shown). In our hands, the best amount of starting material for a good quality RNA and DNA, accomplishing the requirements for RNA sequencing (RNAseq) and Whole Genome Bisulphite Sequencing analysis (WGBS), was 300 galls and 1000 RCs per biological replicate at 3 dpi and 250 galls and 500 RCs at 14 dpi. The best extraction method was using a mortar and a pestle as shown in Portillo et al. (2006). Taking into consideration the difficulty in collecting the material, we have also measured the weight of a single RC of approximately 1 cm length (Figure 1, Steps 1-3; Collection of plant material) from Arabidopsis in order to have a reference (0.06 mg). 150 galls and 500 RC at 3 dpi are enough to obtain around 5.8 and 5.7 µg of total RNA from galls and RC, respectively, and 1.1 and 1.3 µg of genomic DNA from galls and RC, respectively. This amount of total RNA is suitable to perform some expression analysis as quantitative-realtime-PCR (qRT-PCR; Supplementary Figure S1), yet we do not recommend using this protocol from less than 100 galls and 300 RC as RNA and DNA yield is variable and DNA is hardly detected from small samples. However, in order to have enough DNA and RNA for downstream high throughput procedures and later validation, we recommend at least 1000 pieces of RCs and 300 hand dissected 3 dpi galls.

Disruption and Homogenization
RNA and DNA are purified from galls and RCs using the AllPrep R DNA/RNA/miRNA Universal Kit from QIAGEN R GmbH (Hilden, Germany) customized protocol (as detailed in AllPrep R DNA/RNA/miRNA Universal Handbook) with several adaptations. These include the disruption, homogenization, RNase A-treated DNA and elution; as this protocol is originally optimized for animal tissue and human whole blood, but not for plant tissue.
Complete disruption of galls and roots are performed using a mortar and pestle. The mortar and pestle are firstly cooled down using liquid nitrogen. When the liquid nitrogen is almost evaporated, the galls or root segments from uninfected plants are placed on the mortar using a sterile spatula and the tissue is grinded thoroughly using the pestle. 300 µL of RLT Plus Buffer (QIAGEN R ) supplemented with 2-mercaptoethanol (10 µL per mL of RLT Plus Buffer) is added to a liquid nitrogen-frozen mortar with the biological sample and grinded until forming an homogenous paste. When thaw, the lysate is transferred to a 2 mL microcentrifuge tube and the tissue left on the mortar walls is recovered with 50 µL of the same buffer. The lysate is pipetted directly into a QIAshredder R spin column (QIAGEN R ) and centrifuged for 2 min at 18000 × g (centrifugal force). The supernatant is transferred, without disturbing the pellet, to an AllPrep R DNA Mini spin column (QIAGEN R ) and centrifuged for 30 s at 18000 × g. RNA extraction (from the flow-through) is performed immediately and the column containing the DNA is kept in the fridge at 4 • C (Figure 1, Steps 4-12; Disruption and homogenization).

RNA Extraction Procedure
For RNA extraction, the customized protocol proposed by the manufacturer is followed using the upper aqueous phase ) and ethanol, following manufacturer's instructions. To elute the RNA, 30 µL RNase-free water is added directly to the spin column membrane and centrifuged for 1 min at 9000 × g and this step is repeated at least a second time using a new microcentrifuge tube (Figure 1, Steps 13-34; Total RNA purification).

DNA Extraction Procedure
The spin column kept at 4 • C is washed with Buffer AW1 (QIAGEN R ) and incubated with a mix of 20 µL Proteinase K (QIAGEN R ) and 0.6 µL RNase A (100 mg/mL, QIAGEN R ) in Buffer AW1 (QIAGEN R ) for 5 min at room temperature. Consecutively, washes with Buffer AW1 and AW2, following manufacturer's instructions, are carried out and the genomic DNA is eluted by adding 100 µL of elution buffer. This step is repeated twice. The three tubes containing 100 µL of eluted DNA each are placed in a vacuum concentrator at room temperature at 40-60 kPa for 6 h, until the volume is reduced approximately to 30 µL. The three eluates are pooled in the same tube if the concentration of the first elution is not sufficient for the experiment (Figure 1, Steps 35-46; Genomic DNA purification).

Concentration and Quality of Genomic DNA and Total RNA
Concentration and purity of genomic DNA and total RNA is assessed in a spectrophotometer (NanoPhotometer Classic R , Implen, Munich, Germany), using a 3 µL aliquot of the total solutions (Figure 1, Step 47; Assess DNA and RNA concentration and ratios). DNA and RNA purity is estimated from the A 260 /A 280 and A 260 /A 230 ratios obtained (Figure 2) and by gel electrophoresis of 100-150 ng of RNA and 50-100 ng of DNA (Figure 3).
DNA integrity is checked through electrophoresis (Figure 1, Step 48; Integrity of RNA and DNA by gel electrophoresis), using an agarose 1% gel. A high molecular weight size band and a very light smear below the bands is indicative of high genomic DNA integrity.
RNA integrity is evaluated with an Agilent 2100 Bioanalyzer using the RNA Bioanalyzer Pico 6000 chip (Agilent Technologies, Inc., Santa Clara, CA, United States). In order to obtain the electropherograms, 1 µL of total RNA solution from each sample is used (Figure 1, Step 49; Integrity of RNA by electropherogram). The ratio of the peak areas (25S/18S), corresponding to the 25 and 18S ribosomal RNA (rRNA), the RNA Integrity Number (RIN) and the presence of peaks representing small size RNA bands, are used to assess RNA integrity (Figure 4).
( 2)   . From left to right -Black bars represent the total amount of genomic DNA or RNA, as indicated, from 300 galls at 3 dpi to 250 galls at 14 dpi. White bars represent the total amount of genomic DNA or RNA, as indicated, extracted from 1000 segments of control uninfected root tissue equivalent to galls at 3 dpi and 500 equivalent to galls at 14 dpi.
FIGURE 3 | Integrity assessment by gel electrophoresis of total RNA and genomic DNA extracted from galls at 3 days post infection (dpi) and 14 dpi and uninfected control root samples. From the left to the right: samples loaded were RNA and DNA from galls (G) and control roots (RC) samples either at 3 dpi or at 14 dpi, as indicated. A high molecular weight genomic DNA band (>10000 bp) is indicated by a white arrow. Two ribosomal bands are distinguished in the RNA samples and a low molecular weight band, corresponding to the small RNAs represented by an asterisk below the 200 bp marker band ( * ). 1% Agarose gels were stained with 6% ethidium bromide; run at 70 V for 40 min for DNA and 30 min for RNA. The gel of DNA samples at 14 dpi was run at 100 V for 30 min. (a) Thermo Scientific TM GeneRuler TM 1 kb Plus DNA Ladder; (b) 1 kb (+) DNA Ladder; bp, base pairs.

Reagent Setup
All buffers (AW1, AW2, FRN, RLT Plus, RPE) and the RNase-Free DNase (including Buffer RDD) should be prepared previously accordingly to the manufacturer's protocol

Equipment Setup
(1) Mortars and pestles (are previously cleaned with pure bleach overnight, abundantly rinsed with distilled water and then autoclaved): prepare a recipient with liquid nitrogen, which will be used to cool down the mortars and pestles during the extraction.

STEPWISE PROCEDURES
The all in one protocol presented here allows the simultaneous extraction of genomic DNA and total RNA, including small RNAs from plant root tissues. As the major procedures during this protocol are related with the extraction and purification of nucleic acids, including RNA, the use of gloves during all stages is recommended. To avoid the presence of RNases we recommend the use of sterile tubes, a clean benchtop and a solution to clean your benchtop, pipettes and other material such as RNaseZap TM RNase Decontamination Solution, as well as aerosol filter pipet tips, especially for RNA to avoid RNase contaminated aerosols. A fume hood should be used at least in the extractions steps where 2-mercaptoethanol is used. The procedure for the extraction and purification of nucleic acids should not take longer than 3 h. In any case, it always depends on the amount of samples used.

STAGES OF THE PROTOCOL
The presented protocol is divided in four main stages including: (A) collection of plant material; (B) disruption and homogenization of the plant tissue; (C) total RNA purification; (D) genomic DNA purification; and (E) assessment of DNA and RNA concentration, quality and integrity ( Table 1).

STAGE A. COLLECTION OF PLANT MATERIAL
Time: Depending on the type and quantity of plant material During this stage, all materials and reagents should be cleaned and sterilized beforehand. Be cautious in order to make sure that all material and reagents are RNase and DNase-free.
(1) Use RNaseZap TM or similar to clean all equipment, including the stereomicroscope and the tube racks.
(2) When the biological material comes from in vitro culture, it is recommended to drain the excess of water before handling the biological material.

Disruption
(4) Cold down the mortar and pestle using liquid nitrogen. (5) When the liquid nitrogen is evaporated, place the sample on the mortar and grind thoroughly using the pestle (a spatula can be used to help placing the tissue on the mortar). (6) Add 300 µL of Buffer RLT Plus (QIAGEN R ) to the mortar and grind thoroughly using the pestle in order to get a paste while thawing.

KEY NOTES TO KEEP IN MIND DURING STAGE B
(i) A small recipient (for instance, 5 mL microcentrifuge tubes) can be used to cool down the mortar and pestle by pouring the liquid nitrogen on them. If a high volume of liquid nitrogen is added to the mortar at once, the biological sample can be splashed out of the mortar. For this reason, it is also important to add the tissue only when all liquid nitrogen is evaporated. It is also possible to cool the mortar by immersion. (ii) In the case the mortar is too cold, the buffer might get frozen. Start grinding only when a white paste is present and until is thawed. (iii) After the homogenization, a pellet might be formed at the bottom of the collection tube. Do not disturb it.

STAGE C. TOTAL RNA PURIFICATION
Time: approximately 1 h All the following steps are based on the manufacturer's protocol provided by QIAGEN GmbH (Hilden, Germany) in its AllPrep R DNA/RNA/miRNA Universal Kit's Handbook. As proposed by the manufacturer, the initial steps' volumes were adjusted accordingly to the amount of the starting material.
(13) Add 90 µL chloroform to the flow-through from step 12, coming from the homogenization steps, and vortex. Then, centrifuge it using the refrigerated centrifuge at 4 • C for 3 min at 18000 × g in order to separate the phases. RNase-free water and using another microcentrifuge tube for a second/third elution.

KEY NOTES TO KEEP IN MIND DURING STAGE C
(i) Be careful in step 14 not to mix the phases. If it occurs, in order to get clean purified RNA, you might centrifuge it again to get a sharper separation of organic and aqueous phases. (ii) In accordance to the manufacturer's instructions (QIAGEN R ), between steps 26 and 28, the flow-through from step 27, which is enriched in small RNAs (sRNAs), can be applied again in the spin column and centrifuged in order to enrich the total RNA samples with sRNAs. Alternatively, the eluate of step 27 could be kept as it will be enriched in sRNAs. (iii) In the case that the concentration of the first eluate is low, several eluates can be combined together in the same tube. In some instances, the second/third elution can show lower A 260 /A 280 ratios than the first elution. (iv) After extraction, the RNA should be kept on ice for further processing or stored at −80 • C for later studies in order to preserve its integrity.

STAGE D. GENOMIC DNA PURIFICATION
Time: approximately 25 min As for the RNA purification, all the following steps are based on the manufacturer's protocol provided by QIAGEN GmbH (Hilden, Germany) in its AllPrep R DNA/RNA/miRNA Universal Kit's Handbook. In the present protocol for DNA purification, the use of RNase and the optimized elution procedure represent the major changes from the manufacturer's protocol.

KEY NOTES TO KEEP IN MIND DURING STAGE D
(i) The mixture from step 37 should be prepared in a separate microcentrifuge tube, vortexed and centrifuged before being applied to the spin column membrane. In this step, the RNase incubation is performed to avoid RNA contamination.
(ii) It is important that the Buffer EB (QIAGEN R ), from step 44, is preheated to 70 • C before the final elution. It should be maintained in the thermoblock between elutions. (iii) In the case that the concentration of the first eluate is low, several eluates can be combined together in the same tube and concentrated in a speed vacuum concentrator. In some instances, the second and the third elution can show lower A 260 /A 280 ratios than the first elution. (v) After extraction, the DNA should be kept on ice for further processing or stored at −20 • C to preserve its integrity.  (Figure 4).

KEY NOTES TO KEEP IN MIND DURING STAGE E
(i) It is important to check either the A 260 /A 280 and A 260 /A 230 ratios given by the spectrophotometer. An A 260 /A 280 ratio of 2.0 is accepted as indicative of highly pure RNA. An A 260 /A 280 ratio of 1.8 is accepted as indicative of highly pure DNA. Pure nucleic acids usually show an A 260 /A 230 ratio of around 2-2.2. (ii) It is accepted that pure RNA in the electropherograms show a RIN higher than 6.5, the closest to 10 the best, and a 25S/18S rRNA ratio close to 2 or higher. Also, peaks of low molecular weights, indicative of degradation products, should not be present, expect for the population of sRNAs. (iii) Be careful not to run the gel electrophoresis with your RNA samples for a long time as the RNA integrity might be influenced by the high temperatures reached during the electrophoresis. (iv) The electrophoresis cell and combs should be cleaned previously with a normal soap-based detergent and rinsed with distilled water, in order to eliminate any residues and/or nucleic acids-degrading enzymes.

RESULTS AND DISCUSSION
Here, we present a protocol that allows the simultaneous extraction of DNA and total RNA with high quality and enough yield for high throughput sequencing purposes (Figure 1) from the same biological sample of M. javanica-induced galls and its respective control root segments. Due to the banning of nematicides (Ec No1107/2009, there is an urgent need to search for new control strategies. The study of the mechanisms governing the plant-nematode interaction could bring some light on tools as, e.g., genes or epigenetic signatures that could be used in a future as biotechnological tools. In this respect, an emerging topic coming from the molecular analyses combining transcriptomics with cell biology, such as laser microdissection of giant cells of different plant species, is that early developing GCs are characterized by widespread, but specific, gene repression that includes many microRNAs (Barcala et al., 2010;Portillo et al., 2013;Cabrera et al., 2016;Medina et al., 2017). However, the gene silencing mechanism/s involved is/are not completely understood. One hypothesis is that this gene repression is partially mediated by sRNAs either directly (by RNA interference) or indirectly by RdDM. Studies to reveal important clues on the mechanisms behind this gene repression should consider holistic approaches combining genome wide analysis and gene expression such as the methylome, RNAseq, sRNAseq, etc., of nematode feeding sites compared to uninfected tissues. Subtle differences on epigenetic processes are frequent among independent biological replicates of the same tissue and they may not correlate with changes on the mRNAs or sRNAs population also obtained from different replicates of the same tissue. Therefore, the method described here allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample (from Figures 1-4).
We optimized different steps from the manufacturer's protocol including the amount of the starting material, the volumes used in the disruption, the elution steps and the use of RNase during the DNA purification. For the homogenization, we have also included the QIAshredder R spin column, which is given as an alternative for this step by the manufacturer. DNA and RNA suitable for library construction (Figures 5, 6), for sequencing, and for qRT-PCR analyses (Supplementary Figure S1) and possibly other molecular analysis, were obtained from around 250-300 M. javanica galls induced in Arabidopsis roots and 500-1000 control root segments. The DNA and RNA yields ranged from 2 to 2.7 µg; 3.2 to 4.2 µg; respectively for galls at 3 dpi and from 2.8 to 4.1 µg; 12.2 to 18.3 µg; respectively for 14 dpi galls (Figure 2). In all cases A 260 /A 280 ratios were around 2 for both 3 and 14 dpi samples (Figure 2). However, in some cases, the A 260 /A 230 ratio obtained was quite low. Some contaminants might contribute to this low ratio, such as salts, carbohydrates or phenol among others. According to the manufacturer of the Kit used, the A 260 /A 230 ratio of an RNA sample can be reduced even when guanidine thiocyanate, within the extraction buffer, is present at submillimolar concentrations (von Ahlfen and Schlumpberger, 2010). However, the authors claim that in their study, the concentrations of guanidine thiocyanate up to 100 mM in an RNA sample did not compromise the reliability of RT-PCR (von Ahlfen and Schlumpberger, 2010). In our hands, qRT-PCR or libraries for RNAseq were not altered even in the case of this low ratio in the samples used (Supplementary Figure S1 and Figure 5, respectively).
It is important to point that DNA and RNA of good quality could be obtained from lower amount of biological  samples as mentioned in the section "optimization of the starting material, " but, in our hands, nucleic acids obtained from lower amount of tissue did not accomplish requirements for high throughput sequencing, particularly, enough DNA yield for the analysis and later validation. Representative electropherograms of 14 dpi galls show the high quality of RNA obtained either from galls or their control tissues (Figure 4) as RIN numbers were never lower than 9.4 and most of them close to 10 in all samples and replicates. In addition, the 25S/18S ratio values ranged from 2.2 in replicate 3 of RC (RC3) to the highest value in replicate 1 of galls (G1; 2.7) indicative of a high RNA integrity. Accordingly, no peaks corresponding to low molecular weight RNAs, indicative of partial degradation, were detected in the electropherograms (Figure 4). However, a major peak was observed between 25 and 200 base pairs (bp) that is likely due to an enrichment of sRNAs in the total RNA (Figure 4). This is in agreement with the gel electrophoresis which shows that the ribosomal RNA was intact with no evident smear and also a prominent band just below the ladder band of 200 bp, presumably corresponding to the sRNAs peak observed in the electropherograms (compare Figure 3 and Figure 4). As explained in the "Key notes to keep in mind during stage C, " an intermediate step can be performed to enrich the sample in small RNAs. However, even if omitted, some of those small RNAs are still present in the sample (Figure 3, 14 dpi; and Figure 4). Additionally, the 3 dpi gall RNA was used to amplify a gene by qRT-PCR, the CHROMOMETHYLASE 2 (CMT2), and its corresponding normalizer, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C2 (GAPC2). Clear bands of 60 and 86 bp for CMT2 and GAPC2, respectively, were observed (Supplementary Figure S1) and reproducible in all 3 technical replicates used, indicating that the RNA obtained is suitable for qRT-PCR analysis.
In respect to the DNA quality, the gel electrophoresis indicated that a high molecular weight band was present in all samples either at 3 dpi or 14 dpi (Figure 3). The incubation with RNase during the genomic DNA purification (Stage D) allowed us to obtain RNA-free genomic DNA as bands of low molecular weight were not detected (Figure 3).
After construction of the RNA and DNA libraries for RNAseq and WGBS, electropherograms show a fragment size distribution with a major peak around 300 bp in the RNA libraries and around 360 bp in the DNA libraries (Figures 5, 6) indicative of good quality libraries for subsequent RNAseq and WGBS, respectively. Additionally, the genomic DNA extracted and purified with this method was also appropriate to use in an ELISA based Kit (MethylFlash TM Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric), EpiGentek) to measure the global DNA methylation state of galls and control roots. Both independent biological and technical replicates gave reproducible results (data not shown).
Although, the use of this protocol has led us to reliable results, during the course of the protocol optimization, we have encountered several problems (see Table 2 for troubleshooting). The main limiting problem was the amount of starting material. For that, as mentioned above, we weighted the material and optimized the minimum amount needed for the extraction. When the amount of starting material was low, the quantity and quality of the DNA and RNA was affected, which could lead to deficient library construction. During the optimization, we have also used different disruption methods as a TissueLyser II (QIAGEN R ) and a mortar and pestle. The second option, in addition of being cheaper and consequently available in all laboratories, resulted more efficient (data not shown), as mentioned also by Portillo et al. (2006). Therefore, with the changes introduced, this protocol could be also helpful to obtain high quality nucleic acids from other Arabidopsis tissues, and could be easily extended to other structures produced after the infection of other plant-parasitic nematodes, as well as other plant-pathogens.
Available methods to extract and purify nucleic acids from plants are numerous, yet, this is the first time that a simultaneous extraction and purification of both genomic DNA and total RNA from the same biological sample of galls and plant roots of high quality and suitable for current high throughput sequencing methods, is described. The importance of this protocol relies on the fact that obtaining RNA and DNA of high quality from the same biological sample is a great advantage for studies combining gene expression and epigenetics. Therefore, it is particularly helpful, to study changes in the genomic DNA leading to changes in gene expression as the cells of the tissues used for RNA and DNA extraction are exactly the same as well as their cellular status.

CONCLUSION
The method herein described was successfully adapted for the simultaneous extraction of nucleic acids, genomic DNA and total RNA, from the same biological sample. Here we describe the case of the M. javanica galls formed in Arabidopsis roots as compared to control root segments. Understanding the mechanisms leading to changes in gene expression during the formation and maintenance of the feeding sites induced by these pathogens, nowadays entangle combined studies at the transcriptomic and epigenomic level. In this context, the importance of this protocol relies on the fact that obtaining RNA and DNA of high quality from the same biological sample is a great advantage for the study of epigenetic alterations in the DNA, related to changes in gene expression within the same tissue. One of the main reasons is that the cellular status of the tissue used for RNA and DNA extraction is exactly the same as they are obtained simultaneously. Therefore, we believe that this method can be also extended for the simultaneous extraction of high quality RNA and DNA from other Arabidopsis tissues or other structures induced by other plant-parasitic nematodes or even possibly other pathogens/symbionts interactions.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files.

AUTHOR CONTRIBUTIONS
AS, VR-F, ÁM-G, and MB performed most of the experiments related with the protocol. AS and CE aimed the protocol. VR-F, MB, and CE guided AS for the experiments. AS and CE wrote the manuscript. The final version was supervised by AS, VR-F, CF, and CE. All authors read the manuscript.