eamag/protocols-with-steps huggingface dataset:
TITLE: Gene Expression
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
RT-PCR is a technique to quantify gene expression in samples.

[STEPS]
SECTION: Gene Expression
1. Perform gene expression on tissue samples of the ventral midbrain (containing the substantia nigra pars compacta, SNpc).
SECTION: RNA Extraction
2. At due time points, homogenize tissue samples in 1 mL of QIAzol Lysis Reagent (Qiagen, #79306) using a rotor-stator homogenizer. 
SECTION: RNA Extraction
3. Isolate the total RNA from homogenized tissue samples using RNeasy Lipid Tissue Kit (Qiagen, #74804) including Dnase digestion. 
SECTION: RNA Extraction
4. At the end, re-dissolve RNA samples in 30 µL of RNase-free water and determine their concentrations spectrophotometrically by A260 (Nanodrop-ND 1000). 

SECTION: Reverse Transcription
5. Perform cDNA synthesis using Retroscript Kit (Ambion #1710, Austin, Texas) according the manufacturer’s instructions. 
SECTION: Reverse Transcription
6. Add 50 µL of water solution containing 0.5 µg of each pool to an equal volume of 2X TaqMan Universal PCR Mastrer Mix (Applied Biosystems). 

SECTION: Real Time PCR
7. Perform real-time quantitative PCR using TaqmanTM Assay Reagents on an Step One Detection System (Applied Biosystems) according to manufactures protocol. 

SECTION: Real Time PCR
8. Process tissue samples as above. 
SECTION: Real Time PCR
9. Lyse the cell samples in lysis buffer (Qiagen) and store at -80 °C until the RNA extraction following manufacture’s instructions. 
SECTION: Real Time PCR
10. Remove residual genomic DNA by incubating with DNase I, RNase-free (Qiagen) and elute from the RNeasy mini columns with RNase-free water. 
SECTION: Real Time PCR
11. Quantify the amount of total RNA was using a NanoDrop ND-100 (Nano Drop Technologies) and synthesize the cDNA from 2 µg of total RNA using the Retroscript Kit (Ambion). 
SECTION: Real Time PCR
12. After purification using QIAquick PCR Purification kit (Qiagen), use 250 ng of cDNA for Real-time PCR using pre-developed Taqman Assay Reagents (Applied Biosystems). 

SECTION: Real Time PCR
13. Perform real-time quantitative PCR with Step One Detection System (Applied Biosystems) according to manufacturer protocol. 
 

SECTION: Real Time PCR
14. Using the delta delta Ct (2-ΔΔCt) comparative method, quantification of the abundance of target gene expression was determined relative to β-actin with respect to the control group, express the results as arbitrary units (AU). 
SECTION: Real Time PCR
15. Indicate relative fold changes over WT in the respective treatment groups.
TITLE: Factors associated with admission to the intensive care unit and mortality in patients with COVID-19, Colombia
AUTHORS: Jorge Enrique Machado-Alba, Luis Fernando Valladales Restrepo, Manuel E Machado Duque, Andres Gaviria Mendoza

[DESCRIPTION]
Introduction:Coronavirus disease 2019 (COVID-19) has affected millions of people worldwide, and several sociodemographic variables, comorbidities and care variables have been associated with complications and mortality.
Objective: To identify the factors associated with admission to intensive care units (ICUs) and mortality in patients with COVID-19 from 4 clinics in Colombia.
Methods: This was a follow-up study of a cohort of patients diagnosed with COVID-19 between March and August 2020. Sociodemographic, clinical (Charlson comorbidity index and NEWS 2 score) and pharmacological variables were identified. Multivariate analyses were performed to identify variables associated with the risk of admission to the ICU and death (p<0.05).
Results: A total of 780 patients were analyzed, with a median age of 57.0 years; 61.2% were male. On admission, 54.9% were classified as severely ill, 65.3% were diagnosed with acute respiratory distress syndrome, 32.4% were admitted to the ICU, and 26.0% died. The factors associated with a greater likelihood of ICU admission were severe pneumonia (OR: 9.86; 95%CI:5.99-16.23), each 1-point increase in the NEWS 2 score (OR:1.09; 95%CI:1.002-1.19), history of ischemic heart disease (OR:3.24; 95%CI:1.16-9.00), and chronic obstructive pulmonary disease (OR:2.07; 95%CI:1.09-3.90).The risk of dying increased in those older than 65 years (OR:3.08; 95%CI:1.66-5.71), in patients with acute renal failure (OR:6.96; 95%CI:4.41-11.78), admitted to the ICU (OR:6.31; 95%CI:3.63-10.95), and for each 1-point increase in the Charlson comorbidity index (OR:1.16; 95%CI:1.002-1.35).
Conclusions:Factors related to increasing the probability of requiring ICU care or dying in patients with COVID-19 were identified, facilitating the development of anticipatory intervention measures that favor comprehensive care and improve patient prognosis.

[STEPS]
1.
TITLE: Qiagen- AllPrep DNA/RNA/protein Mini Kit for tissue
AUTHORS: Heather Robeson, Jing Jin, Mohammed S. Orloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol is based on Qiagen-AllPrep DNA/RNA/Protein Mini kit for tissue </div></div>

[STEPS]
?. [Sample disruption and homogenization]
Homogenize the lysate in Buffer RLTDisruption using a mortar and pestle. Homogenization using a QIAshredder homogenizer.
?. [Sample disruption and homogenization]
Centrifuge the lysate for 3 min at full speed. Remove the supernatant by pipetting and transfer the homogenized lysate to an AllPrep DNA spin column placed in a 2 ml collection tube.Centrifuge for 30 s at ≥8000 x g (10,000 rpm).
?. [Sample disruption and homogenization]
Place the AllPrep DNA spin column in a new 2 ml collection tube, and store at room temperature (15–25°C) or at 4°C for later DNA purification. Use the flow-through for RNA purification.
?. [Total RNA purification]
To the flow-through, add 96–100% ethanol: either 250 μl (if 350 μl Buffer RLT was used) or 400 μl (if 600 μl Buffer RLT was used). Mix well by pipetting.
?. [Total RNA purification]
Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (10,000 rpm). Transfer the flow-through to a 2 ml tube for protein purification.
?. [Total RNA purification]
Add 700 μl Buffer RW1 to the RNeasy spin column. Centrifuge for 15 s at ≥8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.
?. [Total RNA purification]
Add 500 μl Buffer RPE to the RNeasy spin column. Centrifuge for 15 s at ≥8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.
?. [Total RNA purification]
Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (10,000 rpm) to wash the spin column membrane.
?. [Total RNA purification]
Place the RNeasy spin column in a new 2 ml collection tube and discard the old collection tube with the flow-through. Centrifuge at full speed for 1 min.
?. [Total RNA purification]
Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μl RNase-free water to the spin column membrane. Centrifuge for 1 min at ≥ 8000 x g (10,000 rpm) to elute the RNA.
?. [Genomic DNA purification]
Add 500 μl Buffer AW1 to the AllPrep DNA spin column. Centrifuge for 15 s at ≥8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.
?. [Genomic DNA purification]
Add 500 μl Buffer AW2 to the AllPrep DNA spin column. Centrifuge for 2 min at full speed to wash the spin column membrane.
?. [Genomic DNA purification]
Place the AllPrep DNA spin column in a new 1.5 ml collection tube. Add 100 μl Buffer EB (preheated to 70°C) to the spin column membrane. Incubate at room temperature (15–25°C) for 2 min, and then centrifuge for 1 min at ≥8000 x g (10,000 rpm) to elute the DNA.
?. [Genomic DNA purification]
Repeat to elute further DNA.
?. [Sample disruption and homogenization]
Excise the tissue sample and determine the amount of tissue.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Provides a short introduction to phylogenetics and how to build a phylogenetic tree from concatenated ribosomal marker proteins. </p>
<p> </p>
<p>Open this protocol inside the virtual machine (details in 'Start Instructions') for easy copy, paste of commands into the command line terminal window. </p>

[BEFORE_START]
<p>Before starting, please visit the ECOGEO website for more information on this "Introduction to Environmental 'Omics" training series. The site contains a pre-packaged virtual machine that can be downloaded and used to run all of the protocols in this protocols.io collection. In addition to the VM, the website contains video and presentations from our initial "Intro to Env 'Omics" workshop held at the Univ. of Hawai'i at Manoa on 25-26 Jul 2016.<br /><br />Please email ‘ecogeo-join@earthcube.org’ to join the ECOGEO listserv for future updates.</p>

[STEPS]
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TITLE: Single cell RNA sequencing of retrogradely labeled mouse stellate ganglion neuron
AUTHORS: Seoeun  Lee, Daniele  Neri, Lori  Zeltser

[DESCRIPTION]
A collection of protocols to sequence and analyze single neurons from the stellate ganglia of mice after injection of Cholera Toxin B (CTB) in the interscapular brown adipose fat.

Step 1: Retrograde labeling of brown adipose tissue (BAT)-projecting sympathetic neurons with cholera toxin B (CTB)

Step 2: Ganglia dissociation and single-cell sorting

Step 3: Single-cell sequencing and analysis

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is kit-free and can be used to simultanously isolate high quality genomic DNA of <em>Symbiodinium</em> and Aiptasia from symbiotic anemones which can be used e.g. as PCR template for genotyping.</p>
<p> </p>
<p> It is based on the method described in Coffroth et al., 1992.</p>

[BEFORE_START]
<p>Recipes:</p>
<p><strong>2×CTAB Buffer</strong></p>
<p><em>(Coffroth et al. (1992) Marine Biology 114: 317-325)</em></p>
<p> </p>
<p><strong>End concentrations:</strong>                                             <strong>For 100 ml buffer mix:</strong></p>
<p>1.4 M              NaCl                                                            28 ml of a 5 M stock solution</p>
<p>20 mM            EDTA (pH 8)                                                 4 ml of a 0.5 M stock solution</p>
<p>100 mM          Tris/HCl (pH 8)                                          10 ml of a 1 M stock solution</p>
<p>2% (w/v)        CTAB powder                                                2 g</p>
<p><u>Add after filter sterilization under a hood:</u></p>
<p>0.2% (v/v)      β-mercaptoethanol (<strong><u>TOXIC!!!</u></strong>)              200 µl</p>
<p> </p>
<ul>
<li>Add ddH<sub>2</sub>O to just under 100 ml.</li>
<li>Warm to 65°C under stirring to bring the CTAB into solution.</li>
<li>Once dissolved, bring final volume to 100 ml using a graduated cylinder.</li>
<li>Filter sterilize (0.2 µm) into sterile 50 ml Falcon tubes and store at −20°C.</li>
</ul>
<p>Heat to 65°C before usage since freezing leads precipitation of the CTAB.</p>
<p> </p>

[GUIDELINES]
<p>Make sure to use Phenol:Chloroform:Isoamyl alcohol (25:24:1) which is buffered with TE to a neutral to slightly basic pH (everthing starting from 6.7 should work) since DNA stability decreases at acidic pH (the opposite is true for RNA).</p>
<p> </p>
<p>Be sure not to exceed the maximal ×g for which your microcentrifuge tubes are certified otherwise they might get damaged during centrifugation and release all the hazards substances (and the DNA of course) into the rotor. You don't want that!</p>
<p> </p>
<p>If making your own stock solutions of RNase A from powder be aware that some products contain DNase impurities. If this should be the case you need to boil the stock solutions once for 15-20 min at 100°C and let cool down at room temperature to inactivate the DNases. </p>

[STEPS]
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TITLE: Tetiaroa Sugar Water Crazy Ant Sampling
AUTHORS: Laura Barragan, clairewiegand, ame

[DESCRIPTION]
Sugar water vial protocol for crazy ant sampling.

[BEFORE_START]
Wear long pants and long sleeve top. 

[STEPS]
1. Place vial at designated point on Gaia and label the transect number on the vial
2. Take photo to capture surroundings

4. After 90 minutes, retrieve vials in the order you placed them
5. Count ants in vial; if there are too many ants in the vial, freeze the vial and recount later
6. Record the number of ants in vial on Epicollect5

3. Repeat step until you run out of vials/you've hit each data point on Gaia
7. Clean out test tubes and throw everything away
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This tutorial covers a full QIIME workflow using Illumina sequencing data and was adapted from a <a href="http://nbviewer.ipython.org/github/biocore/qiime/blob/1.9.1/examples/ipynb/illumina_overview_tutorial.ipynb" target="_blank">tutorial</a> on the QIIME website.. This tutorial is intended to be quick to run, and as such, uses only a subset of a full Illumina Genome Analyzer II (GAIIx) run. We'll make use of the <a href="http://www.ncbi.nlm.nih.gov/pubmed/22134646" target="_blank">Greengenes</a> reference OTUs, which is the default reference database used by QIIME. You can determine which version of Greengenes is being used by running print_qiime_config.py. This will be <a href="http://www.ncbi.nlm.nih.gov/pubmed/22134646" target="_blank">Greengenes</a>, unless you've configured QIIME to use a different reference database by default.The data used in this tutorial are derived from the <a href="http://www.ncbi.nlm.nih.gov/pubmed/21624126" target="_blank">Moving Pictures of the Human Microbiome</a> study, where two human subjects collected daily samples from four body sites: the tongue, the palm of the left hand, the palm of the right hand, and the gut (via fecal samples obtained by swapping used toilet paper). These data were sequenced using the barcoded amplicon sequencing protocol described in <a href="http://www.ncbi.nlm.nih.gov/pubmed/20534432" target="_blank">Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample</a>. A more recent version of this protocol that can be used with the Illumina HiSeq 2000 and MiSeq can be found <a href="http://www.ncbi.nlm.nih.gov/pubmed/22402401" target="_blank">here</a>.

[BEFORE_START]
Make sure you have QIIME installed or are using the QIIME virtual machine.  This tutorial is specifically designed to run on the HPC at the University of Arizona.

[GUIDELINES]
This tutorial is run using the suite of python tools in QIIME.

[STEPS]
?. 
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TITLE: Plasmid: NK588 (pAct-nanoluc)
AUTHORS: David Booth, Nicole King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The attached file is the full sequence for a plasmid that uses gene regulatory elements from </span><span style = "font-style:italic;">S. rosetta </span><span>to drive the expression of nanoluc luciferase upon transfection in </span><span style = "font-style:italic;">S. rosetta</span><span>. </span></div><div class = "text-block"><span>The plasmid is backbone is pUC19 and can be propagated in </span><span style = "font-style:italic;">E. coli</span><span> with Ampicillin selection. </span></div></div>

[STEPS]
?. 
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TITLE: Immunofluorescence and confocal microscopy
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the process of immunofluorescence and confocal microscopy.

[STEPS]
SECTION: Steps
1. Seed the cells on glass coverslips (12 mm #1.5) at a concentration of 100.000 cells/well, and after treatment with Rapalog for the indicated time, fix in 4% paraformaldehyde (28906, Thermo Fisher Scientific) for 10 min at Room temperature.
SECTION: Steps
2. After washing with PBS, permeabilize the cells with 0.1% (v/v) Triton X-100 (9002-93-1, Sigma-Aldrich) in PBS for 5 min.
SECTION: Steps
3. Perform the blocking with blocking buffer (5% (v/v) BSA (9048-46-8, Sigma-Aldrich) and 0.05% (v/v) Triton X-100 diluted in PBS) for 60 min at Room temperature. 
SECTION: Steps
4. Dilute the primary and secondary antibodies in blocking buffer and incubate for 60 min at Room temperature with three PBS washing steps in between. 
SECTION: Steps
5. Mount the cells on microscopy slides in DAPI Fluoromount-G mounting medium (0100-20, Southern Biotech), which stains the nuclei, and store at 4 °C until use. 
SECTION: Steps
6. Perform the confocal microscopy with a Zeiss LSM700 laser scanning confocal microscopy with Plan-Apochromat 40×/1.30 Oil DIC, WD 0.21 mm objective.
TITLE: Real-time quantitative polymerase chain reaction (RT-qPCR)
AUTHORS: An.Huang, Qiaowa Gong

[DESCRIPTION]
Real-time quantitative polymerase chain reaction (RT-qPCR) helps determine the expression level of a certain gene by amplifying DNA according to the target mRNA template. This protocol describes the procedure of conducting RT-qPCR using Promega GoTaq® qPCR Master Mix A6001.

[STEPS]
1. Thaw the GoTaq® Master Mix and Nuclease-Free Water. Thaw the cDNA templates and primer.   
 


3. Determine the number of reactions to be set up, including negative control reactions. Add 1 or 2 reactions to this number to compensate for pipetting error. 
 

4. Prepare the reaction mix (minus DNA template) by combining the GoTaq® qPCR Master Mix, PCR primers and Nuclease-Free Water as described below. The DNA template is added in Step 6. Vortex briefly to mix.  

5. Add the DNA template (or water for the no-template control reactions) to the appropriate wells of the reaction plate. 
6. Seal the tubes or optical plate, and centrifuge briefly to collect the contents of the wells at the bottom. The samples are ready for thermalcycling. 
 

7. Thermalcycle in qPCR equipment. Realtime qPCR is performed with an initial denaturation of 3 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 60°C, and 20s at 72°C.
2. Vortex the GoTaq® Master Mix for 3–5 seconds to mix. Vortex at low speed to avoid aeration.
TITLE: Culture and purification of Plasmodium berghei ookinetes
AUTHORS: Benito Recio Totoro

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A collection of protocols we use routinely in the lab to produce and purify ookinetes of </span><span style = "font-style:italic;">Plasmodium berghei</span><span>. </span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Electrophysiology by MEA. Electrical recording and stimulation of the cultures using MEA set-up (by Multichannel Systems).

[STEPS]
SECTION: MEA set-up preparation
?. 
SECTION: MEA software preparation
?. 
SECTION: MEA electrical stimulation set-up
?. 
SECTION: MEA electrical stimulation software
?.
TITLE: NEBNext Ultra End Prep Mixture E7442
AUTHORS: Isabel Gautreau

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [NEBNext End Prep]
Starting Material: 5 ng-1 μg fragmented DNAMix the following comonents in a sterile, nuclease-free tube:  AB1ComponentVolume2End Prep Enzyme Mix3 µl3End Repair Reaction Buffer (10X)6.5 µl4Fragmented DNA55.5 µl5Total Volume65 µl
AB1ComponentVolume2End Prep Enzyme Mix3 µl3End Repair Reaction Buffer (10X)6.5 µl4Fragmented DNA55.5 µl5Total Volume65 µl
?. [NEBNext End Prep]
Mix by pipetting, followed by a quick spin to collect all liquid from the sides of the tube.
?. [NEBNext End Prep]
Place in a thermocycler, with the heated lid on, and run the following program:30 minutes @ 20°C30 minutes @65°CHold at 4°C
?. [NEBNext End Prep]
Proceed directly to NEBNext Ultra Ligation Module (NEB #E7445).
TITLE: Inoculating a Liquid Bacterial Culture
AUTHORS: Addgene The Nonprofit Plasmid Repository, Joe Kaczmarski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for inoculating a liquid bacterial culture. To see the full abstract and additional resources, visit </span><a href="https://www.addgene.org/protocols/inoculate-bacterial-culture" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/inoculate-bacterial-culture/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. If you haven't already, prepare autoclaved liquid LB. For example, to make  of LB, weigh out the following into a  glass bottle: NaCl Tryptone Yeast Extractadd dH2O to Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminium foil. Autoclave (media setting) and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at .
400 mL
500 mL
4 g
4 g
2 g
400 mL
Note, if your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.
0 Room temperature
?. When ready to grow your culture, add liquid LB to a sterile tube or flask and add the appropriate antibiotic to the correct concentration (see table below in step 9).
Note, if you intend to do a mini-prep you will usually want to start   in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a   Erlenmeyer flask.
?. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
?. Drop the tip or toothpick into the liquid LB + antibiotic and swirl your tube or flask.
In some cases (e.g. ON cultures for minipreps) it is best to not leave the tip/toothpick in the media. Instead, just keep tip on pipette swirl a few times in the media (or pipette up and down a few times) and then remove the tip along with the pipette.
?. Loosely cover the culture with sterile aluminium foil or a cap that is not airtight.
?. Incubate bacterial culture at  for - in a shaking incubator (ideally on ~25° angle) at ~ 250 rpm.
37 °C
Note, some plasmids or strains require growth at  . If so, you will likely need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at lower temperatures.
?. After incubation, check for growth, which is characterized by a cloudy haze in the media (see image below).
Notes: Some protocols require bacteria to be in the log phase of growth. Check the instructions for your specific protocol and conduct an OD600 to measure the density of your culture if needed.A good negative control is LB media + antibiotic without any bacteria inoculated. You should see no growth in this culture after overnight incubation.
?. (Optional) For long term storage of the bacteria, you can proceed with Creating a Glycerol Stock.
?. You can now isolate your plasmid DNA from the bacterial culture by isolating your plasmid DNA.
TITLE: Sucrose Preference
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Test for anhedonia-like behavior in mice.

[STEPS]
1. Individually house mice.
2. Place two bottles in random positions in each cage:
1) one bottle containing regular water
2) the second containing 1% sucrose diluted in water
3. Record the weight of the each bottle before and after 24 hours.
4. Sucrose preference index is defined as the total sucrose consumption over the total liquid consumption.