eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution,in vivo.

[STEPS]
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Droplet Breakage
?. 
SECTION: Reverse Transcription
?. 
SECTION: Exonuclease I treatment
?. 
SECTION: cDNA Amplification
?. 
SECTION: Tagmentation
?. 
SECTION: Sequencing
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei Isolation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Nuclei and beads co-encapsulation
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Droplet Breakage
?. 
SECTION: Reverse Transcription
?. 
SECTION: Reverse Transcription
?. 
SECTION: Reverse Transcription
?. 
SECTION: Exonuclease I treatment
?. 
SECTION: Exonuclease I treatment
?. 
SECTION: Exonuclease I treatment
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: cDNA Amplification
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Tagmentation
?. 
SECTION: Sequencing
?. 
SECTION: Nuclei Isolation
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This tutorial focuses on performing a comprehensive metagenomic analysis from whole-genome shotgun sequencing data for microbiome studies. It is divided in to different steps that use the following metagenomic, computational tools: <strong><a href="http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2066.html" target="_blank">MetaPhlAn</a></strong>, <strong><a href="https://bitbucket.org/nsegata/graphlan" target="_blank">GraPhlAn</a></strong>, <strong><a href="http://genomebiology.com/2011/12/6/R60" target="_blank">LEfSe</a></strong>, and <strong><a href="http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002358" target="_blank">HUMAnN</a></strong>. Our last addition to the PhlAn tools is <strong><a href="http://www.nature.com/ncomms/2013/130814/ncomms3304/full/ncomms3304.html" target="_blank">PhyloPhlAn</a></strong> that can be used in metagenomics for phylgenetically and taxonomically place contigs assembled from whole metagenomic sequencing samples.This tutorial is also a step-by-step description of the metagenomic pipeline we used in our review paper about <a href="http://www.nature.com/msb/journal/v9/n1/full/msb201322.html" target="_blank">computational meta'omics</a> (specifically <a href="http://www.nature.com/msb/journal/v9/n1/fig_tab/msb201322_F4.html" target="_blank">Figure 4</a>).

[STEPS]
?.
TITLE: DNA Extraction of Placenta Tissue 
AUTHORS: Cayla Mason

[DESCRIPTION]
This protocol describes extracting DNA from placenta tissue, starting with dissociation.

[STEPS]
SECTION: Tissue Dissociation and Lysis
1. Place placenta tissue samples in screw cap tubes with O-ring gaskets on ice to thaw.
SECTION: Tissue Dissociation and Lysis
2. Label a 1.5 mL microcentrifuge tube for each placenta sample. Aliquot 1 mL silica beads per tube.
SECTION: Tissue Dissociation and Lysis
3. Add 540 uL Buffer ATL and 20 uL Proteinase K to each tissue. Pour in prepared beads.
SECTION: Tissue Dissociation and Lysis
4. Tightly close tubes. Place tubes in Bead Beater,  and tightly close screw-on parts. Homogenize for 2 minutes. 
SECTION: Tissue Dissociation and Lysis
5. Incubate lysates with beads in 56C water bath or heat block for 2 minutes.
SECTION: DNA Extraction
11. Thaw lysates at room temperature.
SECTION: Tissue Dissociation and Lysis
7. Add 600 uL Buffer AL to screw-cap tubes with beads. Vortex and quick spin. Transfer supernatant to microcentrifuge tube with lysate. Discard screw-cap tube with beads.
SECTION: Tissue Dissociation and Lysis
6. Transfer lysate to pre-labeled microcentrifuge tubes. Do not discard beads.
SECTION: Tissue Dissociation and Lysis
8. Vortex lysate until homogenized.
SECTION: Tissue Dissociation and Lysis
9. Incubate lysates 56C for 10 minutes. Quick spin.
SECTION: Tissue Dissociation and Lysis
10. Freeze at -80C or proceed with DNA extraction.
SECTION: DNA Extraction
12. Prepare columns and buffers. Add 25 mL 100% ethanol to Buffer AW1 and 30 mL 100% ethanol to Buffer AW2.
SECTION: DNA Extraction
13. Add one-half volume (approximately 500 uL) 100% ethanol to lysate. Mix thoroughly by vortexing.
SECTION: DNA Extraction
14. Pipet maximum 700 uL of the mixture to column. Centrifuge at >=6000g for 1 minute. Discard flow through. Repeat this step until all lysate has passed through column.
SECTION: DNA Extraction
15. Add 500 uL Buffer AW1. Centrifuge for 1 minute at >=6000g. Discard flow-through and collection tube.
SECTION: DNA Extraction
16. Place the spin column in a new 2 mL collection tube. Add 500 uL Buffer AW2. Centrifuge for 3 minutes at 20000g. Discard flow-through and collection tube.
SECTION: DNA Extraction
16.1. While spinning, label 1.5 mL microcentrifuge tubes for elution. 
SECTION: DNA Extraction
17. Transfer column to pre-labeled microcentrifuge tubes.
SECTION: DNA Extraction
18. Elute DNA by adding 100 uL Buffer AE or nuclease-free water to the center of the membrane. Incubate for 1 minute at room temperature. Centrifuge for 1 minute at >=6000g. 
SECTION: DNA Extraction
19. Repeat step 18.
SECTION: DNA Extraction
20. Store eluted DNA at -20C.
TITLE: Coronal cryosectioning of mouse brains
AUTHORS: Lauren C. Faget, Thomas Hnasko

[DESCRIPTION]
Cryostat coronal cryosectioning of mouse brain tissue

[BEFORE_START]
PFA-perfused, 30% sucrose-cryoprotected, and flash-frozen mouse brains are stored at -80C until cryosectioning

[STEPS]
SECTION: Procedure
1. Retrieve frozen mouse brain stored at -80 °C.

SECTION: Procedure
2. Place brain in cryostat. Set chamber temperature to -16 °C to -20 °C. Set brain holder temperature to -20 °C. Let brain equilibrate to temperature for at least 20 min before sectioning.

SECTION: Procedure
3. Insert chuck and blade into cryostat.
SECTION: Procedure
4. Apply a drop of OCT on the chuck, and affix brain vertically (olfactory bulbs up).
SECTION: Procedure
5. Apply another layer of OCT at the base of the brain, turning the brain-holder with the other hand. Cover the cerebellum and the junction between the cerebellum and cortex with OCT. Allow OCT to freeze solid (~5 min).

SECTION: Procedure
6. Cut 16 to 30 micron sections. Adjust angle of medio-lateral and ventro-dorsal axis so that sections are free of cutting artifacts, symmetrical, and well-aligned to reference atlas. 
SECTION: Procedure
7. Collect free-floating sections in 48-well plate.
SECTION: Procedure
8. Use parafilm to seal the lid to the plate (air-tight) and refrigerate at 4 °Cuntil further processing.

SECTION: Procedure
6.1. Use of anti-roll plate is optional and can help reduce rolling of sections, but must be carefully adjusted to avoid cutting artifacts.
SECTION: Procedure
6.2. For immunohistochemistry we typically cut 30-micron sections for best results. For RNAscope we typically cut 16 to 20-micron sections.
SECTION: Procedure
7.1. Label your plate lid and base with brain/experiment details using lab marker and/or lab tape. 
SECTION: Procedure
7.2. Pre-fill wells with 1 mL 1X PBS containing 0.01% sodium azide (100mg/L). 

SECTION: Procedure
7.3. Can use syringe with blunt needle to collect sections after sectioning. Dip needle into PBS and collect section by gently touching it on the side of the section. 
SECTION: Procedure
7.4. Collect 5 sections per well. Or for experiments that require finer resolution of coordinate location can collect sections serially, adding 1 section per well until the end of the plate and start back at well #A1.
TITLE: Membrane Tube Image Analysis
AUTHORS: Liv  Jensen

[DESCRIPTION]
This protocol details Membrane Tube Image Analysis.

[STEPS]
SECTION: Membrane Tube Image Analysis
1.  In FIJI, create new image stacks from two rectangular selections: one containing a section of the membrane tube, and one containing the approximately horizontal section of the guv.
SECTION: Membrane Tube Image Analysis
2. Save these new stacks as .tif files and paste paths into enrichment quantification script.
SECTION: Membrane Tube Image Analysis
3. Run enrichment quantification, adjusting parameters such as ktub to fit your experimental conditions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol details how to perform read recruitement to the contigs generated from the previous protocol using Bowtie2. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Protocol name 
AUTHORS: Joanna Sendecka

[DESCRIPTION]
This is a test protocol created for the purpose of developing guidelines.

[STEPS]
SECTION: Section one
1. Here we start writing the protocol. It will last 10 min

SECTION: Section one
1.1. and here we continue
SECTION: Section one
2. We give examples of amounts e.g. volume 0.5 L or weight 200 g or voltage 3 V

SECTION: Section one
3.
TITLE: Colorimetric LAMP/RT-LAMP Protocol
AUTHORS: Felipe Navarro Martínez, Fernan Federici

[DESCRIPTION]
This protocol describes how to perform colorimetric LAMP / RT-LAMP reactions with homemade buffers, together with home-brewed BstLF and MMLV enzymes. 

It is based on previous protocols of our lab (see LAMP/RT-LAMP Reaction protocol), but since two of the most common indicators used in colorimetric LAMP (phenol red and neutral red) require weakly buffered reactions, the buffer composition needed to be changed. This was done according to indications by Poole et al (2017).

The colorimetric methods rely on the inherent proton production by DNA polymerases during the amplification process, which leads to a drop in the reaction pH that can be followed using these pH indicators. The results can be seen directly with the "naked eye", without the need for equipment or hands-on processing (Tanner et al., 2015).

  


[STEPS]
SECTION: 2X Buffer Preparation
1. Prepare 1000 µL of 2X Colorimetric Buffer Mix according to the following table:

 
Two main differences compared to non-colorimetric LAMP reaction are the addition of dNTPs in the buffer, as they influence the reaction pH, and the removal of Tris-HCl as this component is vital to maintain the pH of the buffer at a stable point, which is not desirable for pH-dependent detection
The optimal concentration of KCl for home-brewed BstLF is 50 mM (see LAMP/RT-LAMP Buffer protocol)

SECTION: LAMP / RT-LAMP
4. Prepare the LAMP or RT-LAMP reaction mix on ice according to the following tables:
SECTION: 2X Buffer Preparation
2. Measure the reaction pH with strips and adjust pH using NaOH 250 mM to a final value of pH 8-9.
 

3. Add nuclease-free water to a final volume of 1000 µL.  Make aliquots and store them at 4 °C until use.
3. 
SECTION: LAMP / RT-LAMP
5. Incubate the reactions at 65ºc for 35 minutes and observe results with the naked eye.
 
 
SECTION: LAMP / RT-LAMP
5. Vortex and spin reaction mix. 
Always keep reactions and components on ice to reduce spurious amplification risk or sample degradation.

SECTION: LAMP / RT-LAMP
6. Incubate the reaction at 65 °C for 35 min
 


SECTION: LAMP / RT-LAMP
7. Visualize results with the naked eye.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Ten healthy male volunteers took 5 g dose of either TJ-100 or lactose without knowing the identity of the substance. Each volunteer underwent two MR examinations after taking the other substance (TJ-100 or lactose) on separate days; they drank 1200 mL tap water and underwent cine MR examinations after 10 min. A steady-state free precession sequence was used for imaging, which was performed three times at 0, 10, 20, 30, 40, and 50 min. Bowel contraction frequency and distention score were assessed. Wilcoxon signed-rank test was used, and differences were considered significant for <em>P</em> values &lt;0.05.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: The role of multiprotein bridging factor 1 (MBF1c) in Funariaceae species under salt stress
AUTHORS: Maria Victória Magalhães de Vargas, Guilherme Afonso Kessler de Andrade, Sara Navarrete Bohi Goulart, Bruna Mota Bernardes, Filipe Victoria

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Bryophytes are one of the most successful plants in nature and can tolerate several environmental stresses, such as saline stress. MBF1 is a transcription factor that may be involved in reprogramming transcription, which in turn may confer tolerance to stress conditions. The objective of this study was to quantify PaMBF1c under saline stress in three species of moss:</span><span style = "font-style:italic;">Funaria hygrometrica</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">Physcomitrium acutifolium</span><span>, and</span><span style = "font-style:italic;">Physcomitrella patens</span><span>. All moss species underwent 12 h and 24 h treatment with salt at different concentrations, followed by qRT-PCR experiments.</span></div><div class = "text-block"><span>In </span><span style = "font-style:italic;">F. hygrometrica</span><span>, the MBF1c gene was up-regulated when subjected to 12 h salt treatment at a medium concentration, and may be an important factor for the tolerance of plants to NaCl concentrations. In</span><span style = "font-style:italic;">P. acutifolium,</span><span>24 h NaCl treatment at of 400, 600, and 800 mM resulted in a gradual up-regulation of MBF1c expression, reflective of the increasing NaCl concentration. In</span><span style = "font-style:italic;">Physcomitrella,</span><span>the expression profile of MBF1c showed a down-regulation when treated with 200, 400, and 800 mM NaCl, suggesting that MBF1c may not interfere with the saline stress response in this plant.</span></div><div class = "text-block">The diversity of these expression profiles demonstrate that the MBF1c response is not universal neither among plants nor between species of the same family, as shown in the mosses studied.</div></div>

[STEPS]
TITLE: A Simple RNA Preparation Method for SARS-CoV-2 detection by RT-qPCR
AUTHORS: Aniela Wozniak, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Ariel Cerda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages. </div></div>

[STEPS]
TITLE: Isopropanol DNA Precipitation - Best for DNA Concentration
AUTHORS: Lynn    Doran

[DESCRIPTION]
Protocol to precipitate extracted DNA from an aqueous solution to increase concentration or resolubilize in a different storage buffer.

*Use isopropanol DNA precipitation if your DNA is suspended in a very large volume, if your DNA concentration is low, or you are trying to concentrate large molecular weight DNA fragments and remove smaller fragments.

*Use ethanol DNA precipitation if you are trying to remove salt contamination or precipitate small DNA fragments.

Additional Resources:
New England Biolabs, "DNA Precipitation: Ethanol vs. Isopropanol". June 23, 2015
Green, Michael R. and Joseph Sambrook, "Precipitation of DNA with Isopropanol". doi:10.1101/pdb.prot093385Cold Spring Harb Protoc2017.
Qiagen, "How can I precipitate genomic DNA using isopropanol?".

[GUIDELINES]
DNA precipitation performed at room temperature minimizes co-precipitation of salt that interferes with downstream applications.

[STEPS]
1. Transfer desired amount of aqueous DNA solution to a fresh microtube. (70 ul)
2. Add 3.0 M pH 5.2 sodium acetate to the DNA solution to result in a final concentration of 0.3 M sodium acetate. (7 ul)
3. Add 0.6–0.7 volume of isopropanol at room temperature and mix well. (50 ul)
4. Point caps of tubes out to help identify pellet location below cap. Centrifuge the sample immediately at 10,000–15,000 x g for 20–30 min in a microcentrifuge tube at 4°C.

5. Identify the pelleted DNA. Carefully decant the supernatant fluid into a fresh labeled tube without losing the pellet. 
 

6. Wash the DNA pellet by adding 500 ul or more (depending on the size of the preparation) of room-temperature 70% ethanol. Flick tube and invert several times until pellet is removed from the side of the tube and all sides can be washed.
 

7. Centrifuge the sample at 10,000–15,000 x g for 20–30 min at 4°C.
8. Visually identify the pellet. Pour out ethanol.  Tap tube gently on paper towel, being sure to keep track of the pellet.
9. Optional: For high quality DNA applications requiring very clean DNA extractions, repeat steps 6-8. More ethanol washes will increase quality but will negatively affect recovery.
10. After last ethanol wash, use a pipette to remove any ethanol that remains after decanting.
11.  Allow samples to air dry at room temperature for 10-20 min. When pellet turns from white to clear the pellet is dry.  Do not over dry.
 


12. Resuspend the DNA pellet in Tris, TE (pH 8.0), or molecular grade water.  Incubate in 4 °C overnight for more complete resuspension.  For long term storage, store at -20 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol decribes how to plate bacteria in order to isolate a single colony of bacteria. Once this protocol is completed the isolated single colony can be used directly for experimental purposes or used to build up a freezer stock of the bacterial species. 

For further information on building up a freezer stock, continue onto the protocol 'Building up a freezer stock of bacteria'  by the same author on completion of this protocol.

[BEFORE_START]
Before starting this protocol users will need to have prepared LB broth and LB agar plates (or alternatively a genus specific media plate, i.e. Pseudomonas isolation media). If instruction is required for making LB broth and/or LB agar please see the protocols 'LB (Luria-Bertani) liquid medium' and  'LB (Luria-Bertani) agar'  by the same author.

[GUIDELINES]
For efficient bacterial growth in liquid media, the media should not come above the widest area of the conical shaped flask/tube. Chose a flask/tube wisely depending on the volume of media which you plan to inoculate. For a 10 mL inoculation, a 50 mL falcon tube is recommended.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Calcium imaging in astrocytes
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Calcium imaging in astrocytes 

[STEPS]
1. Coat a 10 cm plate with 0.1% gelatin for 20 min 

11. Plate 50k astrocytes of infected astrocytes 
2. From a 80% confluent 10 cm plate of astrocytes obtained from protocol  https://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2
3. Add 3 mL of   for 5 min 

4. 300 rcf, 3 min, 25 °C 

5. Add pTALV-fUBIGW-GCAMP8SIRES-PURO lentivirus 1/100 in   to the pellet and resuspend the cells

6. Incubate  5 min 

7. Add to a gelatin coated 10 cm plate with a 9 ml   warmed media.

8. 2880 min  observe fluorescence levels to confirm infection

9. Wait 10080 min  for the GCAMPs expression to reach stable levels

10. Coat a 2 cm plate with 0.1% gelatin for 20 min 
12. after 2880 min  change to mature astrocyte media

13. Before the start of imaging aim for 80% confluency.
14. Place the plate on a 
 
plate holder.
15. Continuously perfused with ACSF with the following composition (in mM): NaCl 125, KCl 5, D-Glucose 10, HEPES-Na 10, CaCl2 3.1, MgCl2 1.3.  using  a  ValveBank8 II controller.
16. Using a 10X objective. GCaMP8s was excited using a 480 nm (Mic-LED-480A, Prizmatix), an HQ480/40x excitation filter, a Q505LP dichroic mirror, and an HQ535/50m emission filter (Semrock).
17. Sample at a rate of 4.7 fps with a frame exposure of 200 ms at 160x120 pixels (4x4 binning).
18. ROI segmentation of GCaMP8s, raw fluorescence extraction, and background correction can be performed with Nikon Elements software.