eamag/protocols-with-steps huggingface dataset:
TITLE: qPCR assay for detecting Triturus cristatus
AUTHORS: Alexander Eiler, Omneya Osman

[DESCRIPTION]
eDNA assay to detect great crested newt Triturus cristatus

[GUIDELINES]
Laboratory work space and equipment were sterilized by UV-light and DNase solution and 70% ethanol. Filter pipet tips were used in all steps of the laboratory work.
 Negative controls of DNase/RNase free water were used in each qPCR assay. 

[STEPS]
SECTION: DNA extraction
1. 
DNA extraction was performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was estimated using Nanodrop.  Qiagen DNeasy power water sterivex kit: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
SECTION: DNA extraction
2. Internal control 

When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified with the sample DNA and can be detected as a positive control for the extraction process. 

http://www.primerdesign.co.uk/assets/files/internal_control_handbook_dna.pdf?timestamp=1469446474

SECTION: DNA extraction
3. Primers

 

SECTION: DNA extraction
5. PCR mixture
 

SECTION: DNA extraction
6. Amplification conditions

 

SECTION: DNA extraction
4. Standard dilution
DNA of Triturus cristatus was serially diluted from 1e2-1e-4 for qPCR experiment. 
Triturus vulagaris was also tested as related species.
SECTION: DNA extraction
7. qPCR was performed in BioRad qPCR machine CFX96.
 


SECTION: DNA extraction
8. Internal PCR control. The Cq value obtained with the internal control will vary significantly depending on the extraction efficiency, the quantity of DNA added to the PCR reaction, and the individual machine settings. 
Cq values of 27±3 are within the normal range.
TITLE: RT-qPCR
AUTHORS: Karyna Tarasova, Sinan Gültekin, Angkana Kidtiwong, iris.gerner Gerner, Florien Jenner

[DESCRIPTION]
Gene expression levels by RT-qPCR

[STEPS]
SECTION: RT-qPCR
1. Phase separation
SECTION: RT-qPCR
1.1. For gene expression analysis adherent cells were lysed using a mixture of TRIzol reagent (Invitrogen, Thermo Scientific, Germany) + 1% β-Mercaptoethanol (Sigma-Aldrich, USA) (100µL/per well in 96 well plate), snap frozen and stored at -80°C until further processing. After thawing, 200 µL of chloroform (Honeywell, US) were added to achieve for phase separation (centrifugation 13,000g at 4°C for 15min). 

SECTION: RT-qPCR
1.3. The obtained RNA pellet was washed and centrifuged (5 minutes at 13,000g at 4°C) twice with ice-cold 75% EtOH and dissolved in 20 µL of Nuclease-free water (Invitrogen, Thermo Scientific, Germany). 
SECTION: RT-qPCR
2. For the qPCR reaction, RNA samples at a concentration of 1 ng/µL were used. The RevTrans QPCR One-Step EvaGreen kit (Bio&Sell, Feucht, Germany) was used for cDNA synthesis and the subsequent qPCR reaction according to the user manual. The reaction mixtures were incubated for 15 minutes at 50°C for cDNA generation, followed by the qPCR reaction: 95°C for 5 minutes, 95°C for 15 seconds, 55°C for 20 seconds and 72°C for 30 seconds. For each gene, a reaction mixture without the total RNA template was run as a negative control. 

SECTION: RT-qPCR
2.1. The transcript data was analyzed using Agilent AriaMx 1.1 software (Agilent Technologies, Santa Clara, California, USA). The transcript level for the genes of interest was normalized to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and acidic ribosomal phosphoprotein P0 (RPLP0) and presented as a logarithmic fold change to the healthy condition.
SECTION: RT-qPCR
2.2. All primers were designed using Primer3 software. Specificity of the primers was analysed using the NCBI primer blast tool and in silico PCR tool with the UCSC genome browser.
SECTION: RT-qPCR
1.2. The upper phase was collected into a new eppendorf and RNA precipitation was performed by the addition of isopropyl alcohol (Sigma-Aldrich, USA) and glycerol (Thermo Scientific, Germany). The resulting mixture was incubated for 15 minutes on ice and then centrifuged for 15 minutes at 13,000g at 4°C. 
SECTION: RT-qPCR
1.4. Genomic DNA was removed using a DNase treatment and removal kit (Invitrogen, Thermo Scientific, Germany) following the user manual.
3.
TITLE: Full genome PCR amplification of all African Hepatitis B Virus genotypes
AUTHORS: Louis S IV Le Clercq, Sheila Mary Bowyer, Simnikiwe Mayaphi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This method is and adaptation of the full genome amplification protocol published by Gunther </span><span style = "font-style:italic;">et al.</span><span>, able to amplify most known genotypes. The primers bind near the fold in the genome where the DNA is partially double-stranded. Since genotype A1 of HBV frequently has two point mutations in the reverse primer binding region, a second primer was designed to circumvent them when amplifying A1 isolates. This protocol was used to amplify specimens from occult infections of HBV with genotypes A, D, and E.   </span></div></div>

[STEPS]
?. [Master Mix preparation]
Prepare the following two mixes  :
on ice
?. [Master Mix preparation]
Master Mix 1:For one  reaction combine the following:dNTPs25 mM200 uM0.2 uLP115 uM300 nM0.5 uLP2 or P2_A115 uM300 nM0.5 uLExpand Hi Fi Buffer10x1x2 uLMgCl215 mM1.5 mM-ddH2On.a.n.a.11.8 uL
15 µl
dNTPs25 mM200 uM0.2 uLP115 uM300 nM0.5 uLP2 or P2_A115 uM300 nM0.5 uLExpand Hi Fi Buffer10x1x2 uLMgCl215 mM1.5 mM-ddH2On.a.n.a.11.8 uL
?. [Master Mix preparation]
Master Mix 2:For one  reaction combine the following:Expand Hi Fi Buffer10x1x0.5 uLExpand Hi Fi Enzyme3.5 U/uL2.6 U0.75 uLddH2On.a.n.a.3.75 uL
5 µl
Expand Hi Fi Buffer10x1x0.5 uLExpand Hi Fi Enzyme3.5 U/uL2.6 U0.75 uLddH2On.a.n.a.3.75 uL
?. [Master Mix preparation]
Add  to   for a   reaction in thin walled PCR tubes.At the Thermal cycler, add an additional  to each tube after initial denaturation.
[Master Mix 1]
[template DNA]
20 µl
[Master Mix 2]
?. [Thermal cycling]
Perform thermal cycling according to the following conditions:Initial denaturation at   for  2 minutes.Cooling to   before adding Mix 2.       Denaturation at   for 40 seconds       Annealing at   for 90 seconds       Elongation at   for 180 seconds*       *Add 180 seconds every 10 cyclesCooling/hold at
94 °C
58 °C
94 °C
55 °C
68 °C
4 °C
?. [Electrophoresis]
Perform TBE-gel electrophoresis to confirm success of amplification prior to amplicon clean-up.
?. [Electrophoresis]
Prepare a   gel with  and  , pre-stained with  .* is usually added at a concentration of   per  .
[Stock]
[TBE]
?. [Electrophoresis]
Load of PCR product to the gel using   such as  and molecular weight marker, e.g.  , and run at  for  .
5 µl
[loading buffer]
100
?. [Electrophoresis]
Visualize and capture gel on an appropriate imager and paired software, e.g. the
?. [Electrophoresis]
Expected result:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This single-tube library construction protocol is for degraded DNA using BGISeq-500 sequencing platform.</p>

[BEFORE_START]
<p>Calculate the DNA input and prepare the library adapters.</p>

[STEPS]
?. 
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?. 
?.
TITLE: Chromosomal DNA extraction from Gram-positive bacteria
AUTHORS: Anders Kiledal, Julia Maresca

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Extraction of high-molecular-weight DNA from Gram-positive bacterial species, with optional steps for removing surfactants. This DNA is suitable for sequencing and the protocol can be scaled up at least 5-fold. Modified from a protocol by Tina Wecke, </span><a href="http://2012.igem.org/wiki/images/6/6b/LMU-Munich_2012_Isolation_of_genomic_DNA_from_Bacillus_%28for_PCR....%29.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">LMU-Munich</span></a><span>. </span></div></div>

[STEPS]
?. [Grow culture]
Inoculate 10 mL rich medium from a fresh overnight culture, and incubate at appropriate temperature on shaker. At OD600 of ~0.8-1.0, harvest cells by centrifugation (10 min., 5000 rpm).
?. [Cell lysis]
Resuspend cell pellet in 2 mL TEN (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl).
?. [Cell lysis]
Add 100 μL lysozyme (20 mg/mL) and incubate for 20 min at 37ºC.
?. [Cell lysis]
Add 20 μL RNAse (10 mg/mL) and incubate for 3 min at 65ºC.
?. [Remove surfactants (optional)]
IF THE STRAIN PRODUCES A SURFACTANT THAT INTERFERES WITH THE PHASE SEPARATION, Add 0.1 volume 3 M sodium acetate and 1 volume cold isopropanol, mix, and incubate on ice for 20 min. Centrifuge for 10 min at 5000 rpm and decant the supernatant. Then resuspend in 400 μL TEN and 550 μL TEN* and transfer to a microcentrifuge tube.
?. [DNA precipitation]
Collect DNA by coiling on the end of a glass Pasteur pipet.
?. [DNA precipitation]
Air dry, then resuspend DNA in 100 μL sterile water overnight at 4ºC.
?. [Phenol & chloroform:isoamyl alcohol extractions]
Add 900 μL phenol, mix by inversion. Centrifuge for 5 min. at 13000 rpm and transfer the upper phase to a clean microcentrifuge tube.
?. [Phenol & chloroform:isoamyl alcohol extractions]
Re-extract once with phenol (1 volume) and twice with chloroform: isoamyl alcohol (24:1 v/v, 1 volume)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for making two&nbsp;250 ml cultures of&nbsp;electrocompetent cells

[BEFORE_START]
Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).

[STEPS]
?. 
?. 
?. 
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?. 
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?.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Media preparation:</p>
<p>1M KH<sub>2</sub>PO<sub>4</sub> 100ml → 13,61g</p>
<p>1M K<sub>2</sub>HPO<sub>4</sub> 100ml → 17,42g</p>
<p>1M Na-Acetate 100ml → 13,61g Trihydrate</p>
<p>1M Acetic acid 100ml → 60,05g (liquid)</p>
<p>1M Glycine 100ml → 7,51g</p>
<p> </p>
<p>Autoclave KH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub> and Na-Acetate and sterilfiltrate Glycine, Acetic acid should be sterile yet</p>
<p> </p>
<p>LB2,5:</p>
<p>25g/l LB broth (Miller)</p>
<p>15g/l NaCl</p>
<p> </p>
<p> </p>
<p>BHIv2:</p>
<p>37g/l Brain heart infusion broth</p>
<p>204 mM NaCl</p>
<p>4.2 mM KCl</p>
<p>23.14 mM MgCl<span style="font-size: 7pt;">2</span> </p>

[STEPS]
?. 
?. 
?. 
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TITLE: HMW DNA extraction for diatoms 
AUTHORS: Raffaela Abbriano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for genomic DNA extraction from diatom cell culture without use of phenol/chloroform. Resulting DNA fragments were suitable for nanopore sequencing.</div></div>

[STEPS]
?. [Harvest cells]
Harvest approximately 7 x 107 - 2 x 108 cells from mid-exponential phase by centrifugation (5000xg, 3 minutes)Wash pelleted cells once with 1X PBS (5000xg, 3 minutes) and remove the supernatant
?. [Cell lysis and RNAse treatment]
Resuspend cells in 600 L Nuclei Lysis Solution (Promega #A7941)Freeze in dry ice or liquid nitrogen for 3 minutes and thaw in 65˚C heat block or water bath for 3 minutes (or until completely thawed)Repeat freeze/thaw cycle 3XHeat to 65˚C for 15 minutesLet sample cool to room temperature and add 1.2 μL of 10 mg/ml RNase A (ThermoFisher #ENO531)Incubate for 15 minutes at 37˚C in heat block or water bathLet sample cool to room temperature
Be aware that rapid temperature changes can cause pressure to build up inside epi tubes, causing the lids to pop open. Use safe lock epi tubes or lid locks for the freeze/thaw step.https://us.vwr.com/store/product/4615516/lidlocks-microcentrifuge-tube-locks-sorenson-bioscience
?. [Protein precipitation]
Add 250 μL Protein Precipitation Solution and mix well (Promega # A7951)Centrifuge (16,000xg) for 40 minutes at 4˚CTransfer supernatant to fresh tube
?. [DNA precipitation]
Precipitate DNA by adding 700 μL 100% isopropanol, mix well by inversionCentrifuge (16,000xg) for 30 minutes at room temperatureWash DNA pellet with 600 μL freshly prepared 70% molecular grade ethanolCentrifuge (16,000xg) for 1 minute and remove all ethanol from the pellet (use quick spin and a smaller pipette tip if necessary to remove all residual ethanol)Air dry DNA pellet for 10-15 minutesResuspend DNA pellet in ~25-50 μL TE buffer, depending on pellet size and desired DNA concentrationLet DNA pellet resuspend in TE buffer at room temperature overnight (HMW gDNA takes longer to dissolve)Determine DNA concentration by Nanodrop
After isopropanol precipitation, DNA pellet may appear as a translucent smear on the side of the tube. Pellet becomes more visible (white) following the ethanol wash step.
?. [Gel electrophoresis]
1. Run 100-200 ng DNA on a 0.8% agarose gelExample data:L = ladder (top band = 40kb)1. Phaeodactylum tricornutum2. Thalassiosira pseudonana3. Chaetoceros muelleri
ABCD1SpeciesYield (ng/uL)260/280260/2302Thalassiosira pseudonana1255.81.81.863Phaeodactylum tricornutum673.81.852.074Chaetoceros muelleri1066.51.891.49
TITLE: GUV preparation
AUTHORS: Liv  Jensen, Chunmei   Chang

[DESCRIPTION]
GUV preparation for membrane tube assay application

[STEPS]
1. GUV Preparation 
1.1. Clean the coverglass.
1.2. Coat cleaned coverslips with 100 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore).
1.3. Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
1.4. Spread a lipid mixture with a molar composition of 70% DOPC, 20% DOPE, 5% DO-PI(3)P, 5% DOPS, 0.3% Atto647N DOPE, 0.01% PEG2000-biotin-DSPE at 1 mg/ml uniformly onto the PVA film.
1.5. Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
1.6. Use 100 μL 320 mOsm sucrose solution for swelling for 1 h at room temperature
1.7. Harvest the GUVs and use them with 12 h.
TITLE: Genome assembly (Nanopore and Illumina reads)
AUTHORS: Rafael Rodrigues Ferrari, Thiago Mafra Batista

[DESCRIPTION]
This protocol offers detailed, step-by-step instructions for students and researchers to assemble nuclear genomes using long reads generated by Nanopore technology. Before assembling the genome, we will align the reads against a bacterial genome database to eliminate potential contamination. The assembled contigs will then be polished using Illumina short reads.

[STEPS]
SECTION: SEQUENCING QUALITY CHECK
1. ****LongQC (https://github.com/yfukasawa/LongQC)****

***Prepare a .pbs file to run the analysis remotely on Sagarana***
 

SECTION: CROSS-SPECIES CONTAMINATION FILTERIN
2. ****Magic-BLAST (https://ncbi.github.io/magicblast/)****

***Index the database***
 
***ONT whole-genome sequencing***

**Prepare a .pbs file to run the analysis remotely on Sagarana**
 
***Illumina whole-genome sequencing***

**Prepare a .pbs file to run the analysis remotely on Sagarana**
 
***Convert output file***
 

SECTION: GENOME SIZE ESTIMATION
3. ****Jellyfish (https://github.com/gmarcais/Jellyfish)****

***Counting k-mers***

**Prepare a .pbs file to run the analysis remotely on Sagarana**
 
**Size estimation**

/////STRATEGY #1: GenomeScope (on my PC)\\\\\

*Go to the directory where reads.histo is located*
 
/////STRATEGY #2: R (on my PC)\\\\\

*Go to the directory where reads.histo is located*
 

SECTION: GENOME ASSEMBLY
4. ****NextDenovo (https://github.com/Nextomics/NextDenovo)****

***Prepare an 'input.fofn' file***
 
***Prepare a 'run.cfg' file***
 
***Prepare a .pbs file to run the analysis remotely on Sagarana***
 

SECTION: GENOME ASSEMBLY
4.1. GENOME ASSEMBLY STATISTICS

****scaffolds_stats****

***Compare two runs and include the stats into a .txt***
 
****BBMap (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbmap-guide/)****

#BBMap is part of BBTools (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/)

***Run the script stats.sh using the main output produced by Flye (on my PC)***
 
****BUSCO (https://busco.ezlab.org/)****

**Run BUSCO (using a docker)**

$docker run --rm -e USERID=$UID -u $UID -v /home/rferrari/:/home/rferrari/ -w /home/rferrari/projetos/collaris/BUSCO_run/genome/post_polishment/NextDenovo/run4_RF_final/SRs ezlabgva/busco:v5.2.2_cv1 busco -i /home/rferrari/projetos/collaris/BUSCO_run/genome/post_polishment/NextDenovo/run4_RF_final/SRs/genome.nextpolish.fasta -l hymenoptera_odb10 --augustus_species Apis_mellifera -o run1 -m geno -c 12

#To see list of available reference datasets

$docker run --rm -e USERID=$UID -u $UID -v /home/rferrari/:/home/rferrari/ -w /home/rferrari ezlabgva/busco:v5.2.2_cv1 busco --list-datasets
SECTION: GENOME POLISHMENT
5. ****NextPolish (https://github.com/Nextomics/NextPolish)****

***Using only short reads***

**Prepare a 'sgs.fofn' file**
 
**Create a 'run.cfg' file**
 
**Prepare a .pbs file to run the analysis remotely on Sagarana**
 
***Using both short and long reads***

**Prepare a 'sgs.fofn' file**
 
**Prepare a 'lgs.fofn' file**
 
**Create a 'run.cfg' file**
 
**Prepare a .pbs file to run the analysis remotely on Sagarana**
TITLE: Slot/Dot Blot protocol.
AUTHORS: beldenwj 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a simple slot/dot blot protocol for detecting antigen on a membrane.  Using the method outline here, one can achieve results in under 90 min because it eliminates the need for running a gel, transfering the protein in a transfer apparatus, blocking, secondary antibody incubation and the washes between primary and secondary antibodies.</div></div>

[STEPS]
?. [Add sample]
1. Apply individual (or pooled) saliva or processed nasal swabs to the slot/dot blot apparatus making note of sample location.2. Turn on the vacuum source so the sample get drawn onto the membrane.3. Disassemble the slot/dot blot apparatus and place the membrane back in 100 % methanol.4. Air dry the membrane for 5 minutes.
?. [Set up Slot/Dot blot apparatus]
1. Assemble 3 sheets of cut GB003 blotting paper pre-wetted with transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol) to the dot blot apparatus.2. Pre-wet a cut sheet of PVDF in 100% methanol for 30 sec and then wash the PDVF in transfer buffer for 2 min.3. Lay the PVDF membrane on the GB003 blotting paper and seal the slot/Dot blot apparatus.
?. [Incubate with HRP-conjugated antibody, wash and develop]
1. Once the membrane is dry, add it to 10 ml of TBST (20 mM Tris, 150 mM NaCl, 0.1% Triton X-100, pH 7.5) (or PBST) containing HRP-conjuated antibody and incubate with gentle shaking 40 minutes.2. Wash the blot 4 times for 5 minutes with 10 ml of TBST3. Add ECL or colorimetric substrate, develop for 2-5 min, and image blot.4. Record the results.
TITLE: A New Method for Quantitative Tandem Mass Spectrometry of Protein Ubiquitination Based on Peptide Isobaric Labeling
AUTHORS: 181830691 , G G, te

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein ubiquitination is a common post-translational modification in organisms, and it participates in many important cellular physiological processes such as the regulation of cell cycle progress, protein degradation and DNA repair. Abnormal protein ubiquitination is closely related to the occurrence and development of many diseases. Qualitative and quantitative analysis of ubiquitination at the proteomic level helps to further understand the process of protein ubiquitination in organisms, and is important for understanding and treating various diseases caused by disorders of the ubiquitination system, especially malignant tumors. significance.</div></div>

[STEPS]
TITLE: Protocol: The efficacy of the Epley maneuver for benign paroxysmal positional vertigo (BPPV) in primary care setting: a systematic review and meta-analysis
AUTHORS: Yusuke  Saishoji, Norio Yamamoto, Takashi Fujiwara, Hideki Mori, Shunsuke Taito

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background:</div><div class = "text-block">Benign paroxysmal positional vertigo (BPPV) is a common disease that is characterized as vertigo associated with a rapid change of the head position. The Dix-Hallpike (D-H) maneuver is considered the gold standard test for the diagnosis of posterior canal BPPV. There is high-quality and compelling evidence that patients diagnosed with posterior canal BPPV should be offered expeditious treatment with Epley maneuver (EM) in Cochrane review 2014. Several randomized controlled trials on the efficacy of EM on BPPV have been published since 2014, but the integrated results of these trials are not yet clear. Furthermore, the efficacy of EM in primary care settings (family doctor or general physician) was not much researched.</div><div class = "text-block">Methods:</div><div class = "text-block"><span>We conducted a systematic review and meta-analysis of randomized sham-controlled trials of the EM for treatment of  posterior canal BPPV. The objective of this systematic review is to clarify the efficacy of the EM for BPPV in primary care settings and subspecialty settings. The outcome of interest is a resolution of vertigo. We will include randomized controlled trials that assess the efficacy of EM. We used a systematic review protocol template(</span><a href="https://dx.doi.org/10.17504/protocols.io.biqrkdv6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.biqrkdv6</span></a><span>). We followed the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 for preparing this protocol</span></div><div class = "text-block">Ethical consideration and dissemination:</div><div class = "text-block">We will only use openly available data. Ethical approval is not necessary. We will publish this systematic review in peer-reviewed journals.</div></div>

[STEPS]
TITLE: Plant material preparation and salt imposition for PlantScreen analysis
AUTHORS: Magdalena Julkowska, Mariam Awlia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of</span><span style = "font-style:italic;">Arabidopsis thaliana</span><span>plants in response to salt stress.</span></div></div>

[STEPS]
TITLE: Tissue freezing in Cryostor solution + processing
AUTHORS: Regina Hoo, Carmen Sancho, Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tissue freezing in cryostor solution and recovery for tissue processing</div></div>

[STEPS]
?. Chill Cryostor solution  (CS10) on ice (C2874-Sigma).
?. Most tissue is collected in Hyperthermosol and kept on ice or at  .
4 °C
?. Place tissue in petri dish, add enough ice cold RPMI/10% FBS to cover the tissue (depend on tissue size).
?. Mince tissue with 2 scapels, just like what we would do for enzymatic digestion.
?. Carefully transfer minced tissue into a 50ml falcon tube.
?. Wash and rinse the petri dish with plenty of RPMI/10% FBS. Transfer the RPMI/10%FBS and remaining tissue pieces into the falcon tube.
?. Centrifuge at 1500 rpm for 3 minutes.
?. Discard supernatant.
?. Resuspend the tissue in 1 ml of ice cod CS10 and transfer into a cryovial.
?. Rinse the falcon tube with 0.8 ml of extra cold CS10 and transfer into the cryovial.
?. Place cryovial in CoolCell and leave in  freezer overnight.
-80 °C
?. Remove cryovial from CoolCell next day, and store cryovial in designated box in  .
-80 °C
?. To process cryopreserved tissue, thaw cryovials in water bath at  .
37 °C
?. Transfer thawed tissue into a falcon tube containing 20ml warm RPMI/10% FBS.
?. Centrifuge at 500 x g for 4 minutes.
?. Discard supernant. Proceed with preferred tissue digestion protocol