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

[DESCRIPTION]
<p>Non Neutrophil cells are depleted by incubating the sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[GUIDELINES]
<p>This procedure is optimized for the isolation of 10<sup>7</sup> to 1 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p>Application notes: To use this product in magnetic separation columns, a titration of the cocktail/beads should be performed. Optimal concentration for magnetic separation columns is lot-specific. Please contact BioLegend Technical Service (tech@biolegend.com) for further assistance on how to use MojoSort™ Nanobeads in magnetic separation columns.</p>
<p> </p>
<p>Chart Protocol:</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/eppecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/eppecje.jpg" data-ofn="image1 Mojosort Mouse Neutrophil Isolation Kit Protocols.io.JPG" /></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Tissue Sectioning
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol details formalin-fixed paraffin-embedded tissue sectioning on a microtome for subsequent tissue staining.

[STEPS]
SECTION: Tissue Sectioning
1. Clean microtome blade with xylene (to remove paraffin) and then 100% ethanol (to dry).
SECTION: Tissue Sectioning
2. Mount blade on microtome, and tighten screws to hold it in place.
SECTION: Tissue Sectioning
3. Mount paraffin block in holder. Manually move the block near, but not touching the blade.
SECTION: Tissue Sectioning
4. Align the block to the blade in both left-right and top-bottom directions by iterative adjustments
followed by checking the alignment.
SECTION: Tissue Sectioning
5. Once the block is directly apposed to the blade and aligned, switch cutting depth to  .

SECTION: Tissue Sectioning
7. Begin sectioning the block. Grasp the first section lightly. Continue sectioning until you have a
ribbon of sections (~6-10 sections).
SECTION: Tissue Sectioning
8. Use camel hair brush to lightly remove ribbon by brushing from one side to the other. Alternatively, lightly separate the section attached to the block from the remainder of the sections by scoring with a razor blade.
 

SECTION: Tissue Sectioning
9. Transfer the ribbon to a 37 °C water bath. Burst bubbles with a syringe needle. Score between
sections with a razor blade.
SECTION: Tissue Sectioning
10. Gently spring apart sections with forceps and mount them in order on glass slides. Transfer the
slides to an upright rack to allow the water to wick off of the sections before transferring to a
metal rack.
SECTION: Tissue Sectioning
11. Continue sectioning (Steps 6-10) until the block is depleted of tissue.
SECTION: Tissue Sectioning
12. Transfer slides to oven to dry at 42 °C for 120 min or more or 60 °C for 20 min or more. 

SECTION: Tissue Sectioning
6. Hydrate block if needed with ammonia. Dry block after hydration.
TITLE: qPCR Identification Haplosporidium pinnae protocol
AUTHORS: Monserrat López-Sanmartín, Gaetano Catanese, Amalia Grau, Jose Maria Valencia, JRafa García-March, JINavas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Real time PCR to identify </span><span style = "font-style:italic;">Haplosporidium pinnae</span><span> from </span><span style = "font-style:italic;">Pina nobilis </span><span>DNA.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The simple pipeline to examine the natural variation in the coding region of your favourite gene, that you either picked by GWAS or just working on for the entire life and would like to examine what else is there to it. The pipeline described in this protocol is based on the SNP data from Arabidopsis, and so you will miss the variation due to non-Col-0 specific insertions or deletions. Nevertheless, this pipeline will give you an insight in the possible alterations of the coding region of your favourite protein</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Elution of nasal lining fluid collected via nasosorption
AUTHORS: Samuel Montgomery

[DESCRIPTION]
This procedure outlines the materials and processes for eluting nasal lining fluid collected using a Mucosal Diagnostics FX-i nasosorption device. This protocol has been successfully used to elute nasal lining fluid for both DNA extraction for 16s rRNA sequencing & metagenomics, and for proteomics using the SomaScan assay.


[GUIDELINES]
This protocol should be performed in a sterile environment to reduce cross contamination between nasosorption devices, to enable metagnomic DNA extraction and analyses

[STEPS]
SECTION: Making elution buffer
1. Add 0.05 % volume of Tween20 to PBS in a sterile environment to reduce cross-contamination

SECTION: Making elution buffer
2. Store at 4 °C  until required

SECTION: Elution of nasal lining fluid
3. Thaw nasosorption device in cryotube on ice


SECTION: Elution of nasal lining fluid
4. Add 300 µL of elution buffer to a labelled 2mL microcentrifuge tube and place on ice

SECTION: Elution of nasal lining fluid
5. Remove the synthetic absorptive matrix (SAM) from the nasosorption device using sterile forceps by tearing it from the handle and place in the microcentrifuge tube containing elution buffer

SECTION: Elution of nasal lining fluid
6. Vortex the microcentrifuge tube for 30 s 

SECTION: Elution of nasal lining fluid
7. Using a new pair of sterile forceps, remove the SAM from the elution buffer, and place into a spinX column on a new 2mL microcentrifuge tube
SECTION: Elution of nasal lining fluid
8. Add the elution buffer from the first tube onto the SAM in the spinX column
SECTION: Elution of nasal lining fluid
9. Centrifuge the SAM and spinX column at 16000 x g for 30 min at 4 °C 

SECTION: Elution of nasal lining fluid
10. Discard the spinX column and SAM
SECTION: Elution of nasal lining fluid
11. Aliquot the eluate into labelled microtubes, and measure total protein concentration using the assay of your choice (Pierce BCA assay, Qubit protein assay etc)
TITLE: Agar bioplastic (simmered) Ag01
AUTHORS: Clara Davis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Agar bioplastic (simmered) Ag01</div></div>

[STEPS]
?. Mix all of the ingredients together in a pot in the amounts above, and stir. Agar  , Water  and Glycerol
12 g
400 mL
18 g
?. Keep mixing until there are no clumps and it is as dispersed as it’s gong to get. Then heat the mixture to boiling point, and simmer for 15 - 20 min, stirring constantly.
?. Scoop out excess froth with a spoon, and make sure there are no clumps. Carefully pour the mixture onto a surface of acrylic or plastic with a frame.
?. After 4-5  hours lift and let it dry hanging.  How long it takes will depend on the temperature and humidity in the room, and it may take several days (depending on your formulation). You won’t be able to remove the plastic from the drying sheet easily until it is completely dry, so be patient! If your first batch turns out too sticky or slimy, you can try it again with slightly less plasticiser.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is for phospho-Tyrosine western blotting (optimized for detecting phospho FMRP protein after IP)</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Total Starch Enzymatic Digestion 
AUTHORS: Lynn Doran, Amanda P. De Souza

[DESCRIPTION]
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

[BEFORE_START]
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.

[STEPS]
1. Prepare fresh daily 120 U/mL α-amylase  (Bacillus licheniformis) in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 3000 U/mL.  Use C1V1 = C2V2 to calculate the volume of α-amylase and MOPS buffer to use.
 

2. Prepare fresh daily 30 U/mL amyloglucosidase (Aspergillus niger) in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL.  Use C1V1 = C2V2 to calculate the volume of amyloglucosidase  and acetate buffer to use.
 

3. Heat the water bath to 75 °C.

4. Add 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.

5. Vortex to suspend all solids.  Flicking the tube may help dislodge the dried starch pellet.
6. Incubate for 30 min at 75 °C in the water bath.

7. Add another 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
8. Vortex to suspend all solids.
9. Incubate for 30 min at 75 °C in the water bath.

10. Cool down the water bath to 50 °C, tubes can be stored at room temp on the counter while water bath cools.
 

11. Add 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

12. Vortex to suspend all solids.
13. Incubate for 30 min at 50 °C in the water bath.

14. Add another 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

15. Vortex to suspend all solids.
16. Incubate for 30 min at 50 °C in the water bath.

17. Place the tubes in ice to stop the reaction, until cool to the touch.
18. Proceed to total starch (as glucose) quantification by NZYtech GOD-POD method or store the samples at -20°C up to one month.
TITLE: Fresh Frozen / OCT Tissue RNA Quality Evaluation -- University of Minnesota TMCs (ThermoFisher Scientific, Invitrogen - Purelink)
AUTHORS: ThermoFisher Scientific, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
RNA quality evaluation of OCT (Optimal cutting temperature) or fresh frozen blocks/samples.
Completed prior to going through -omics and spatial technologies, sc/snRNAseq if applicable.

[STEPS]
SECTION: Purelink RNA Mini Kit Manual
1.   

SECTION: Purelink RNA Mini Kit Manual
2. Product Information 
Catalog Number: 12183018A
https://www.thermofisher.com/order/catalog/product/12183018A 

or 

Catalog Number: 12183020
https://www.thermofisher.com/order/catalog/product/12183020
SECTION: Purelink RNA Mini Kit Manual
3. Complete:  
Nanodrop for RNA quantification 
RIN score for (Eukaryotic total) RNA qualification
TITLE: Tissue Quality Evaluation for Brain Perfusion/Dissection Specimens
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol provides guidance in the assessment of quality of tissues post perfusion and dissection and applies to all such procedures. Perfusion and dissection quality will be categorized on the following scale: perfect, good, fair, and poor.  This SOP also presents written descriptions, and images, of specific category attributes. 

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Criteria for performing total knee arthroplasty without postoperative intensive care monitoring
AUTHORS: Fabricio Loures, João Henrique Reis, Marcelo de Oliveira Campos, Guilherme Morgado Runco, Luísa Doim Vieira Fonseca, Luciana Ferreira Xavier, Nelson Hiroyuki Miyabe Ooka, Liszt Palmeira de Oliveira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">After reviewing the medical literature, we defined objective criteria to indicate intensive care monitoring after total knee arthroplasty. These criteria were adopted in January 2020 and since then none of the 80 patients who underwent total knee arthroplasty at the Pedro Ernesto University Hospital and who met the criteria required intensive care monitoring.</div></div>

[STEPS]
TITLE: DH5α bacteria transformation protocol
AUTHORS: Yoan Coudert

[STEPS]
?. Thaw bacteria on ice
?. Dispense 50μl in a microtube
?. Add 1 to 5 μl of DNA, and carefully flick the tube to mix cells and DNA
?. Place the mixture on ice for 10 minutes
?. Heat shock at 42°C for 30 seconds
?. Place on ice for 5 minutes
?. Pipette 450 μl of room temperature SOC (or LB) into the mixture
?. Incubate at 37°C for 1 hour. Shake vigorously at 250 rpm.
?. Warm selection plates at 37°C
?. Spread desired volume (150-350 μl) onto selection plate
?. Incubate overnight at 37°C
?. Store at 4°C after incubation of needed
TITLE: Induction of non-selective bulk autophagy 
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.

[STEPS]
SECTION: Nutrient starvation experiments
1. To induce bulk autophagy, starve the cells by culturing them in Hank balanced salt medium
(HBSS, Thermo Fisher) for the indicated time.
SECTION: Nutrient starvation experiments
2. Collect cells by trypsinization, wash them in PBS and lyse them in RIPA buffer for 20 min on ice. Clear the samples by centrifugation at 20000 x g, 10 min, 4 °C. 

SECTION: Nutrient starvation experiments
4. Analyse protein samples by SDS-PAGE and western blot analysis.
SECTION: Nutrient starvation experiments
3. Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Organisms used for DNA transformation experiments were tested for sensitivity to five antibiotics in two different concentrations. Cell culture growing in F2 media with 3% seawater was divided to fifteen 15ml tubes, i.e. three tubes for testing each of the antibiotics: antibiotic was added to two samples to the final concentrations noted in Table 1, the third tube in the set served as a control. The cultures were incubated under 12h light cycle and 20°C and checked by light microscophy 1, 4, 7 and 19 days after the addition of antibiotics.</p>
<p> </p>
<table>
<tbody>
<tr>
<td width="92">
<p>antibiotic</p>
</td>
<td width="66">
<p>puromycin</p>
</td>
<td width="61">
<p>blasticidin</p>
</td>
<td width="62">
<p>geneticin</p>
</td>
<td width="76">
<p>hygromycin</p>
</td>
<td width="66">
<p>zeocin</p>
</td>
</tr>
<tr>
<td width="92">
<p>concentration 1</p>
</td>
<td width="66">
<p>5 μg/ml</p>
</td>
<td width="61">
<p>5 μg/ml</p>
</td>
<td width="62">
<p>200 μg/ml</p>
</td>
<td width="76">
<p>200 μg/ml</p>
</td>
<td width="66">
<p>100 μg/ml</p>
</td>
</tr>
<tr>
<td width="92">
<p>concentration 2</p>
</td>
<td width="66">
<p>10 μg/ml</p>
</td>
<td width="61">
<p>10 μg/ml</p>
</td>
<td width="62">
<p>400 μg/ml</p>
</td>
<td width="76">
<p>400 μg/ml</p>
</td>
<td width="66">
<p>200 μg/ml</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Table 1: Tested antibiotics and concentrations</p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
TITLE: 5N HCl
AUTHORS: Jacquelina.Woods  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation. 

[STEPS]
2. Slowly add 10.4 mL  , or equivalent to the water. 

 

1. Measure 14.6 mL of deionized or ultrapure water into sterile container. 

3. Store at 2-8 Room temperature.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Alveolar macrophages were obtained from the specimens of surgically resected lung tissue. Pieces of lung tissues (~10-30 g) obtained from lung parts resected from patients with treatment-refractory pulmonary tuberculosis. Lung tissue specimens were cut into small pieces and rubbed through a metal screen of sieve with pores of 0.5-2.0 mm diameter in phosphate-buffer saline (PBS, pH 7.4) for separating of cellular suspension containing alveolar macrophages from the closed granulematous-fibrotic tissue. Cellular pellets after centrifugation at 400 <em>g </em>for 5 min at room temperature were placed to 24-well plates (Orange Scientific, Belgium) with cover glasses in the bottom and cultured in 0.5 ml complete growth RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine and 50 µg/ml gentamicin (BioloT, Russia) for 16-18 hours at +37 °C in atmosphere containing 5% CO<sub>2</sub>.</p>

[STEPS]
?.
TITLE: Genomic DNA Extraction from Sorted Cells
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This protocol details the extraction of genomic DNA from cells.

[GUIDELINES]
The DNeasy columns cannot take more than 5 x 106 cells per column. Larger pellets can be split into multiple extraction reactions, then combined after elution.

[STEPS]
SECTION: Extraction of genomic DNA from cells
1. Prepare a lysis master mix of the following reagents and mix by vortexing briefly:

 
 
SECTION: Extraction of genomic DNA from cells
2. Aliquot 322 µL of this master mix into labeled 1.7-mL microcentrifuge tubes.
SECTION: Extraction of genomic DNA from cells
3. Resuspend the pellets in 100 µL DPBS for every 5 x 106 cells in each sample pellet.

If you have 10 x 106 cells in your pellet, resuspend in 200 µL, then split into 2 separate tubes.
SECTION: Extraction of genomic DNA from cells
4. Add resuspended cells in 100 µL DPBS to an appropriately labeled tube containing the lysis master mix. Mix by vortexing.
SECTION: Extraction of genomic DNA from cells
5. Incubate the resuspended cells at 56 °C with agitation at 1500 rpm, 30 min.
SECTION: Extraction of genomic DNA from cells
6. Spin down the lysis tubes briefly, then add 200 µL of 96-100% ethanol to the lysed cells. Mix thoroughly by vortexing. Spin the tubes down to remove drops from lids.
SECTION: Extraction of genomic DNA from cells
7. Transfer the lysed cell mixture to a labeled DNeasy mini spin column in a 2mL collection tube (both provided in the kit).
SECTION: Extraction of genomic DNA from cells
8. Centrifuge at 6000 x g, 1 min. Discard the flow-through and collection tube.
SECTION: Extraction of genomic DNA from cells
9. Place the spin column in a new 2mL collection tube and add 500 µL Buffer AW1 (provided in kit) to the column. Centrifuge at 6000 x g, 1 min. Discard the flow-through and collection tube.
SECTION: Extraction of genomic DNA from cells
10. Place the spin column in a new 2 mL collection tube and add 500 µL  Buffer AW2 (provided in kit) to the column. Centrifuge at 20000 x g, 3 min. Discard the flow-through and collection tube.
SECTION: Extraction of genomic DNA from cells
11. Place the spin column in  a new 2mL collection tube and centrifuge at 20000 x g, 1 min.
SECTION: Extraction of genomic DNA from cells
12. Place the spin column in a new (labeled) 1.7-mL microcentrifuge tube. 
SECTION: Extraction of genomic DNA from cells
13. Add 100 µL of molecular grade water to the spin column and incubate at Room temperature for 2 min. Centrifuge at 6000 x g, 1 min. 
SECTION: Extraction of genomic DNA from cells
14. Repeat Step 13 with an additional 100 µL of molecular grade water.
SECTION: Extraction of genomic DNA from cells
15. Transfer any split samples (pellets with more than 5 x 106 cells) into labeled 2.0mL microcentrifuge tubes.
SECTION: Extraction of genomic DNA from cells
16. Quantitate with the Nanodrop, and store the samples at -20 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Calcium imaging - recording the calcium dynamics of cultures using fluorescent calcium dye, fluorescence microscopy and a camera.

[STEPS]
SECTION: Calcium imaging set up
?. 
SECTION: Wash culture before insertion of calcium dye
?. 
SECTION: Insertion of calcium dye
?. 
SECTION: Wash culture after incubation
?. 
SECTION: Recording
?. 
SECTION: Pharmacology - chemical appliacation
?. 
SECTION: Electrical stimulation
?.
TITLE: 70% Ethanol Fixation
AUTHORS: Arnold  Federico

[DESCRIPTION]
This protocol is specifically written for fixing/permeabilizing suspension cell culture in a final volume of 1mL. For DNA content analysis, such as for cell cycle analysis, a precipitating fixative such as ethanol is preferred over formaldehyde fixation (Darzynkiewicz et al. 2001). The process of fixation preserves the cell state, makes cells permeable and allows for storage for extended periods of time.

[STEPS]
SECTION: Protocol
1. Transfer 1 * 106 to 5 * 106 suspended cells to appropriate centrifuge tube and pellet by spinning at 300g for 5 minutes

SECTION: Protocol
2. Discard supernatant without disturbing pellet. Resuspend and wash cells in 1mL 1X PBS with gentle pipetting. Spin down cells again at 300g for 5 minutes. 
SECTION: Protocol
2.1. While cells are spinning, add 700uL of 100% EtOH (pre-cooled in -20ºC)  to new 1.5mL microcentrifuge tube and keep on ice
SECTION: Protocol
3. Discard supernatant without disturbing pellet. Resuspend pellet in 300uL 1X PBS and gently pipette or vortex to break up cell clumps. (Cell aggregates will be stabilized when introduced to ethanol, make sure you have an even cell suspension!).

Transfer all 300uL cell suspension to microcentrifuge tube containing 700uL EtOH on ice and pipette mix to evenly distribute cells.
SECTION: Protocol
4. Transfer cells to 4ºC  for at least 2hr prior to staining/analysis. Cells should be stable in -20ºC for months.
TITLE: Comparative Efficacy of Antidepressants for Symptoms Remission of Gastroesophageal Reflux: A Bayesian Network Meta-Analysis of Randomized Controlled Trails (protocol)
AUTHORS: Xiaobei Si, Deying Bi, Linyu Huo, Yu Lan, Shuo Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objective </span><span>The aim of this study is to compare symptom relieving of antidepressants for gastroesophageal reflux. </span></div><div class = "text-block"><span style = "font-weight:bold;">Methods </span><span>We plan to include randomized controlled trials (RCTs) that evaluate efficacy of antidepressants for gastroesophageal reflux by searching the MEDLINE, EMBASE, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang databases from the starting date of database establishment till September 31, 2019. The words of "gastroesophageal reflux disease", "antidepressant" and their synonyms will be adopted as searching terms. ADDIS 1.16.8 will be used for network meta-analysis, with conduction of node split analysis as well as rank probability analysis. </span></div><div class = "text-block"><span style = "font-weight:bold;">Conclusions</span><span> The present analysis aims to further compare efficacy of symptom relieving of gastroesophageal reflux among antidepressants, which apply clinical evidence for further therapy of gastroesophageal reflux diseases.</span></div></div>

[STEPS]