eamag/protocols-with-steps huggingface dataset:
TITLE: Protocols for Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage‐specific neurons. However, current strategies mainly rely on genome‐integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild‐type proteins. Using these two modified mRNAs with morphogens, we established a 5‐day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA‐induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high‐content neuron cultures for drug discovery. Proteomic analysis of Atoh1‐binding proteins identified the nonmuscle myosin II (NM‐II) complex as a new binding partner of nuclear Atoh1. The NM‐II complex, commonly known as an ATP‐dependent molecular motor, binds more strongly to phosphosite‐modified Atoh1 than the wild type. Blebbistatin, an NM‐II complex antagonist, and bradykinin, an NM‐II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA‐driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM‐II complex as a positive modulator of Atoh1‐driven neuron differentiation. The methodology described here will facilitate the development of mRNA‐driven differentiation strategies for generating iPSC‐derived progenies widely applicable to disease modeling and cell replacement therapy.</div></div>

[STEPS]
TITLE: Culture media for human islets (based ont eh Edmonton protocol)
AUTHORS: Corentin Cras-Méneur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Culture media for human islets</div></div>

[STEPS]
?. [CMRL Media]
of  of  of  of  of
500 mL
8.5 mL
8.5 mL
5 mL
5 mL
TITLE: BHI + v2 salts media
AUTHORS: Matthew Haines

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Vibrio natriegens  </span><span>grows exceptionally well in BHI + v2 salts media (</span><a href="http://2018.igem.org/Team:Marburg/Results" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">link</span></a><span>). Furthermore, this media is routinely used for culturing this organism (Weinstock et al., 2016). However, care must be taken during preperation not to autoclave v2 salts and BHI together. This protocol first generates separate solutions before sterilising and combining them.</span></div></div>

[STEPS]
?. [Prepare stock salt solutions]
Prepare the following salt solutions at the given concentrations:
[NaCl]
[KCl]
[MgCl2.6H2O]
?. [Prepare BHI media]
Dissolve   in   ddH2O in a 1 L graduated bottle.
[BHI dry medium]
400 mL
?. [Sterilise and combine]
Sterilise all solutions by autoclaving.
?. [Sterilise and combine]
Under sterile conditions, transfer the following volumes of stock salt solutions to the BHI media:  ABCD1SaltStock solution (M)Volume (mL)Final concentration (mM)2NaCl520.42043MgCl2.6H2O111.623.24KCl12.14.2
ABCD1SaltStock solution (M)Volume (mL)Final concentration (mM)2NaCl520.42043MgCl2.6H2O111.623.24KCl12.14.2
?. [Sterilise and combine]
Adjust the volume to   using sterile ddH2O.
500 mL
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Widely used protocol to extract DNA from plant leaves.<br />Many versions circulate on the web, this is the version as we use it.<br />It works well on maize, tomato and probably many other plants.

[BEFORE_START]
Prepare all reagents and materials.<br /><br />TE Buffer, pH 8.0 <br />For 250 ml: <br />2.5 ml 1 M Tris HCl, (10 mM) <br />0.5 ml 0.5 M EDTA, (1 mM) <br />Add H2O to 250 ml<br /><br /><br /><br />CTAB Extraction Buffer <br />For 500 ml: <br />10 g CTAB (1.5% w/v) (Cetyltrimethylammonium bromide or Certridium Bromide: C<sub>19</sub>H<sub>42</sub>BrN)<br />50 ml 1 M Tris HCl, pH 8.0 (75 mM) <br />20 ml 0.05 M EDTA, pH 8.0 (100 mM) <br />140 ml NaCl (5M) <br />290 ml H2O

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Kraus et al., 2022 FBXO7 /Park15  
AUTHORS: Felix Kraus

[DESCRIPTION]
The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation, Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: 1) kinetics of pUb accumulation, 2) pUb puncta on mitochondria by super-resolution imaging, 3) recruitment of Parkin and autophagy machinery to damaged mitochondria, 4) mitophagic flux, and 5) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol can be used to prepare nuclear suspensions from fresh or frozen tissue for single cell DNA or RNA sequencing experiments. For RNA analysis it is recommended to use fresh tissue samples, due to RNA degradation that occurs during freeze- thaw cycles, however for DNA analysis either frozen or fresh tissue can be used.  The protocol has been tested on breast tissues, and many frozen tumor tissues including breast, colon prostate and pancreatic samples.
 

[BEFORE_START]
NST-DAPI: Mix 800 ml of NST solution (146 mM NaCl, 10 mM Tris base (pH 7.8), 1 mM CaCl2, 21 mM MgCl2, 0.05% (wt/vol) BSA and 0.2% (vol/vol) Nonidet P-40) with 200 ml of DAPI solution (106 mM MgCl2 and 10 mg of DAPI). Filter-sterilize the solution and store it at 4°C in the dark for up to 1 year. 
Prepare chilled (4°C) Nuclei Wash and Resuspension Buffer: 1X PBS with 1.0% BSA and 0.2U/μl RNase Inhibitor.


[GUIDELINES]
This protocol is for fresh or frozen tissue. 

[STEPS]
SECTION: Tissue preparation
?. 
SECTION: Tissue preparation
?. 
SECTION: Centrifugation
?. 
SECTION: Wash
?. 
SECTION: Wash
?. 
SECTION: Counting nuclei
?. 
SECTION: Counting nuclei
?. 
SECTION: Counting nuclei
?. 
SECTION: Wash
?.
TITLE: Biotin Labeled Nucleotides
AUTHORS: BOC      Sciences

[DESCRIPTION]
Biotin labelled nucleotides, which is a compound linked to nucleotides by the carboxyl site of biotin. If the nucleic acid can be labeled with biotin by enzymatic reaction, the biotin-avidin protein system can be used for tracer detection. Biotin derivatives with long arms, such as N-succinimide biotin esters, are commonly linked to nucleotides to eliminate steric hindrance. Labeled nucleotides can be combined into DNA or RNA fragments by methods such as notch translation, cDNA labeling, and 3T-terminal labeling.
https://bioconjugation.bocsci.com/services/biotin-labeled-nucleotides.html

[STEPS]
TITLE: Opentrons COVID-19 testing: Station A, Zymo kit, 24 or 48 samples
AUTHORS: Max Marrone

[STEPS]
?. [Reagent preparation]
?. [Initial OT-2 setup]
Clean the OT-2.
?. [Reagent preparation]
Pipette Proteinase K from the Zymo kit into a 1.5 mL snap cap tube.For 24 samples, pipette  . For 48 samples, pipette .Place the Proteinase K tube into slot D1 of the reagent tube rack.Prepare the reagent tube rack, an Opentrons 24 tube rack:
110 µl
220 µl
?. [Reagent preparation]
Prepare the reagent tube rack, an Opentrons 24 tube rack.
?. [Reagent preparation]
Pipette Proteinase K prepared from the Zymo Quick-DNA/RNA Viral MagBead kit into a 1.5 mL snap cap tube. The volume depends on how many samples are being processed:  AB1Samples to processVolume of Proteinase K (µL)224110 µL348220 µLPlace the Proteinase K tube in well D1 of the reagent tube rack.
AB1Samples to processVolume of Proteinase K (µL)224110 µL348220 µL
?. [Reagent preparation]
Pipette internal extraction control RNA prepared from the BP Genomics 2019-nCov Detection Assay kit into a 1.5 mL snap cap tube. The volume depends on how many samples are being processed:  AB1Samples to processVolume of internal extraction control RNA (µL)224110 µL348220 µLPlace the internal extraction control RNA tube in well D4 of the reagent tube rack.
AB1Samples to processVolume of internal extraction control RNA (µL)224110 µL348220 µL
?. [Reagent preparation]
?. [Reagent preparation]
Pipette internal extraction control RNA from the BP Genomics kit into a 1.5 mL snap cap tube.For 24 samples, pipette  . For 48 samples, pipette .Place the internal extraction control RNA tube into slot D4 of the reagent tube rack.
110 µl
220 µl
?. [Reagent preparation]
?. [OT-2 deck setup]
Set up labware on the OT-2's deck.
?. [OT-2 deck setup]
In deck slot 7, place the reagent tube rack.
?. [OT-2 deck setup]
In deck slot 3, place a full, sterile Opentrons 20 µL filter tip rack.
?. [OT-2 deck setup]
In deck slot 1, place an empty, sterile NEST 96 deep well plate.
?. [Running the OT-2]
Run the 24- or 48-sample protocol on the OT-2.24 samples: StationA-24samples-Zymo-M-2020-04-13.py48 samples: StationA-48samples-Zymo-M-2020-04-13.py
{"blocks":[{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"90o5i","text":"","type":"unstyled"}],"entityMap":[]}
?. [Running the OT-2]
The output is the NEST 96 deep well plate in deck slot 1. It contains either 24 or 48 wells of .Only the odd-numbered columnns (1, 3, 5, ...) are used. The even-numbered columns are skipped.
[Proteinase K + internal extraction control RNA mix]
TITLE: TRAP Assay
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">The use of total radical-trapping antioxidant parameter (TRAP) has recently been proposed to explore the antioxidant property of a plasma sample. This assay is a measure of oxidative stress in the animals. This protocol describes the procedure used by the DiaComp to measure TRAP.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakis6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakite.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakitn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakitw.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakit6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiue.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakiun.jpg" /></div></div>

[STEPS]
?. [Sample Preparation - DRG:]
1. Remove 2 DRG from vial, cut in half and weigh. Do in duplicate. 2. Add 30 µL 30mM PB and sonicate on 4 on ice. 3. Spin at maximum g’s for 10 min at 4ºC. 4. Remove sup and store on ice.
?. [Performing the Assay:]
1. Using a White Solid Bottom plate, prepare plate by loading buffer for serial dilution. No PB in 1st well, 5 µL PB in following 4 wells and 5 µL in 3 control well. (Control is PB, Luminol and ABAP) (Dilutions, 1:1, 1:2, 1:4, 1:8, 1:16) 2. Prepare ABAP just prior to running assay by dissolving 54.2 mg ABAP in 2 mL PB. (Enough for 3 columns) 3. Load 5 µL sample in wells 1 & 2. Mix the sample & PB in well 2 and remove 5 µL and place in 3rd well with PB and so on. On last dilution discard 5 µL. 4. Prepare Luminol in a 50 mL conical tube and add 200 µL per well. 5. Place plate in Fluoroskan and add 60 µL ABAP per well and press START. 6. Read every 30 seconds for 20 minutes. 7. When reading is done, Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns. 8. Save organized data as an Excel file into the TRAP Assay data folder. Use the naming convention trXXXXXX.xls, where XXXXXX is the date in yymmdd format. *ABAP = 2,2’-azobis(amidinopropane) dihydrochloride *TRAP = Total Radical-trapping Antioxidant Parameter
TITLE: LRRK2 thermal shift assay
AUTHORS: Verena Dederer, chatterjeedeep, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
Thermal shift assay or differential scanning fluorimetry analyzes the effect of small molecules on the thermostability of a protein by gradual heat denaturation and monitoring absorption of the fluorescent dye SYPR Orange at 488 nm.


[STEPS]
SECTION: Fluorescent-based thermal shift assay
1. Prepare 4 µM master mix of protein in buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol) and add 1:1000 dilution of SYPR Orange.
SECTION: Fluorescent-based thermal shift assay
2. Aliquot 20 µL of the master mix into a white 96 well plate.
      
SECTION: Fluorescent-based thermal shift assay
3. Add DMSO or small molecule binder with a final concentration of 10 µM.

SECTION: Fluorescent-based thermal shift assay
4. Seal plate, mix well and centrifuge 30 sec at 500xg.
SECTION: Fluorescent-based thermal shift assay
5. Place plate into MX3005P real-time PCR instrument.
SECTION: Fluorescent-based thermal shift assay
6. Measure fluorescence with excitation and emission filters set to 465 and 590 nm while gradually increase temperature 3K/min during 71 cycles from 25 to 95°C.
TITLE: Measuring tension, pellet transit, and calcium imaging within cell subtypes in response to pelvic nerve stimulation
AUTHORS: Thomas  Gould, Dante  Heredia

[DESCRIPTION]
A protocol to measure the effects of pelvic nerve stimulation on colonic motility

[GUIDELINES]
Nothing beyond steps

[STEPS]
1. After sacrifice, the caudal half of the animal is pinned out.  A ventral midline incision is made and the colon is cut at the cecum and separated from the small intestine, which is removed.  Then the pelvic nerve is dissected by slowly removing all of the muscle bone and cartilage surrounding the distal colon. Leave the tuft of tiisue surrounding the pelvic nerve intact.  For a good reference for the dissection, see: Feng and Gebhart, 2015, J Vis Exp,(95), 52310. The colon plus pelvic nerves are then placed into a sylguard-lined dissection dish filled with oxygenated Krebs-Ringers solution. The colon is then gently flushed of its contents. For all experiments, the colon is perfused with 35-degree, oxygenated Krebs-Ringers for the duration of the experiment.  A suction electrode connected to a 10ml syringe and tubing is then placed over tuft of tissue with pelvic nerve, and upward pressure applied to suck in nerve.
2. For tension, the colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon. An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening. Suture silk is used to connect three force transducers (model TST125C; Biopac Systems, Santa Barbara, CA) to the proximal, transverse and distal segments of the colon. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems). Spontaneous and stimulation-induced responses (20 HZ for 10s) are recorded.
3. For pellet transit, the colon is pinned loosely into a dish and placed under a camera. A mouse fecal pellet that has been dried, then coated with epoxy, and dried again, is placed with forceps into bottom of proximal colon (end of chevrons).The time it takes to begin movement, as well as the speed of movement, through the colon is recorded, both spontaneously and in response to pelvic nerve stimulation at 20 HZ for 10s.
4. For calcium imaging, either Kit-GCaMP6f, ChAT-GCaMP6f, or nNOS-GCaM6f mice are used. In this dissection, the middle colon only is slit from the ventral midline, and pinned with gentle tension. This is the area of the colon that is imaged. Then calcium responses are measured in response to pelvic nerve stimulation at 20 HZ for 10s.
TITLE: Table 2. Multilocus genotyping results of the 24 Giardia-positive samples successfully genotyped at least at one of the three loci investigated. Shushtar County (Iran), 2017‒2018.
AUTHORS: Abdollah Rafiei, Raheleh Baghlaninezhad, Pamela C. Köster, Begoña Bailo, Marta Hernández de Mingo, David Carmena, Esmat Panabad, Molouk Beiromvand

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is designed for use with food-safe materials. It may be used to illustrate principles of gel electrophoresis for children, or as a party novelty for scientifically-minded adults.  It is currently a work in progress and will be updated as necessary. Currently different colors of food dye will run for different times but will not form distinct bands. 

[STEPS]
SECTION: Gelatin Preparation
?. 
?. 
?. 
?. 
SECTION: Food Dye Setup
?. 
?. 
SECTION: Electrophoresis setup
?. 
?. 
?. 
SECTION: Run the gel!
?. 
?. 
?. 
?.
TITLE: Transfection of V5 labelled plasmid derived from regulatory sequences of Blastocrithidia sp. p57
AUTHORS: Binnypreet Kaur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The plasmid named as p57-V5+G418 was cloned in the backbone pBluescript II SK(+).</div><div class = "text-block"><span>The p57-V5+G418 plasmid contains V5-tagged aminoglycoside 3'-phosphotransferase gene (conferring resistance to neomycin) surrounded by regulatory sequences derived from the hexokinase gene of the kinetoplastid </span><span style = "font-style:italic;">Blastocrithidia</span><span>sp. p57 .</span></div><div class = "text-block"><span>We electroporated linearised plasmid (digested with SwaI) into </span><span style = "font-style:italic;">D. papillatum</span><span>cells using an established protocol (Kaur </span><span style = "font-style:italic;">et al</span><span>. 2018) with Amaxa X-001 program and clones were selected under Geneticin resistant marker.</span></div></div>

[STEPS]
TITLE: Use of resources and cost in the management of gastrointestinal hemorrhage caused by oral anticoagulants
AUTHORS: Jorge Machado Alba

[DESCRIPTION]
Introduction: Oral anticoagulants (OACs) used to treat
nonvalvular atrial fibrillation (NVAF) are frequently associated with
complications, especially gastrointestinal hemorrhage (GH).
Objective: To determine the use of resources and costs in
the in-hospital treatment of GH in a group of patients with NVAF from Colombia.
Methods: This was a retrospective study of a cohort of
patients who were diagnosed with NVAF and treated with an OAC (apixaban,
rivaroxaban, dabigatran or warfarin) between 2018 and 2022 and who were
hospitalized for GH. Information on diagnoses, GH location, administered
medications, procedures, hospital stay, and health care costs was collected, adjusted
to 2022.
Results: In total, 59 patients from nine different cities
were identified, the mean age was 82.9 ± 12.6 years, and most were men (61.0%) who
were admitted to nine clinics for GH. High GH was the most frequently
identified form (78.0%). The most commonly used OACs were apixaban (44.1%),
rivaroxaban (35.6%), warfarin (15.3%) and dabigatran (5.1%). The mean hospital
stay was 6.4 ± 5.9 days, and 18.3% of the patients were admitted to the
intensive care unit. Patients also received medications that increase the risk
of bleeding, such as acetylsalicylic acid (45.8%), the antiplatelet inhibitor
P2Y12 (15.3%) and systemic glucocorticoids (23.7%). The average direct cost of
all hospital care was USD 872 ± 1315 per patient, which corresponds to 13% of
the country's gross domestic product per capita.
Conclusions: GH patients with NVAF generates a significant
cost for the use of resources such as medications, procedures and care that
affect the country's health system.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for amplification of the mitochondrial gene "Citocrome Oxidase I" and the nuclear genes "subunit a from the Histone 3" and "Internal Transcribed Spacer 2" from <em>Nephila clavipes</em> (Araneae: Araneidae).</p>

[GUIDELINES]
<p><strong>Mitochondrial gene Citochrome Oxidase I (COI)</strong></p>
<p>Primers: LCO1490 and HCO2198</p>
<p> </p>
<p>Reference: Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. <em>Molecular Marine Biology and Biotechnology</em>, <strong>3</strong>(5), 294–299.</p>
<p> </p>
<p>PCR Reaction: 1μL of template DNA, 1U of Taq DNA Polymerase (Thermo-Fisher), 2.5μL of 10X KCl Buffer (Thermo-Fisher), 2.8μL of 25mM MgCl<sub>2</sub> (Thermo-Fisher), 1μL of 10mM dNTP mix (GE Healthcare), 0.5μL of each forward and reverse primer 10μM, and ddH<sub>2</sub>O to the volume of 25μL.</p>
<p> </p>
<p>PCR cycling: a denaturation step of 3 min at 94ºC, 30 cycles of a denaturation step at 94ºC for 45s, an annealing step at 51ºC for 45s and an extension step at 72ºC for 2 min, and a final extension step at 72ºC for 3 min.</p>
<p> </p>
<p><strong>Nuclear gene for the subunit a of the Histone 3 (H3a)</strong></p>
<p>Primers: H3aF2 and H3aR2</p>
<p> </p>
<p>Reference: Colgan, D.J., McLauchlan, A., Wilson, G.D.F. <em>et al</em>. (1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. <em>Australian Journal of Zoology</em>, <strong>46</strong>, 419–437.</p>
<p> </p>
<p>PCR Reaction: 1μL of template DNA, 1U of Taq DNA Polymerase (Thermo-Fisher), 2.5μL of 10X KCl Buffer (Thermo-Fisher), 2.3μL of 25mM MgCl<sub>2</sub> (Thermo-Fisher), 0.5μL of 10mM dNTP mix (GE Healthcare), 0.5μL of each forward and reverse primer 10μM, and ddH<sub>2</sub>O to the volume of 25μL.</p>
<p> </p>
<p>PCR cycling: a denaturation step of 3-4 min at 94ºC, 30 cycles of a denaturation step at 94ºC for 45s, an annealing step at 46ºC for 1 min and an extension step at 72ºC for 1 min, and a final extension step at 72ºC for 10 min.</p>
<p> </p>
<p><strong>Nuclear intron internal transcribed spacer 2 (ITS2)</strong></p>
<p>Primers: 5.8S and ITS4</p>
<p> </p>
<p>Reference: White, T.J., Bruns, T., Lee, S., Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, <em>et al</em>., eds. <em>PCR Protocols: A Guide to Methods and Applications</em>. New York: Academic Press, 315–322.</p>
<p> </p>
<p>PCR Reaction: 1μL of template DNA, 1U of Taq DNA Polymerase (Thermo-Fisher), 2.5μL of 10X KCl Buffer (Thermo-Fisher), 3.5μL of 25mM MgCl<sub>2</sub> (Thermo-Fisher), 0.5μL of 10mM dNTP mix (GE Healthcare), 0.4μL of each forward and reverse primer 10μM, and ddH<sub>2</sub>O to the volume of 25μL.</p>
<p> </p>
<p>PCR cycling: a denaturation step of 4 min at 95ºC, 30 cycles of a denaturation step at 95ºC for 45s, an annealing step at 61ºC for 45s and an extension step at 72ºC for 2 min, and a final extension step at 72ºC for 10 min.</p>

[STEPS]