eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol 2: MD simulation with Gromacs 
AUTHORS: Phaniendra Alugoju, Vishwambhar Vishnu Bhandare, Vishal S Patil, Krishna Swamy V K D, Prem Kumar Borugadda, Tewin Tencomnao

[DESCRIPTION]
Structural stability of the docked complexes was monitored using MD simulation with Gromacs 2020.5 (RRID:SCR_014565). The protein topology was created using Gromos96 force field and the PRODRG server was utilized to create the topology of the ligand. The docked complexes were solvated using ‘Simple Point Charge’ (SPC) water model in a cuboidal box with adequate size to accommodate the complete complex at the center.The modeled systems were neutralized using required number of counter ions (Na+ / Cl-). The undesirable contacts and steric conflicts were then removed from the neutralized systems using steepest descent followed by conjugate gradient methods for 50,000 steps each. The NVT ensemble used to maintain constant number of atoms, volume, and temperature, further NPT ensemble was used to maintain constant pressure. NVT and NPTensembles were used to maintain temperature. The temperature and pressure were set constant at 300K and 1 bar respectively, during the equilibration of 1ns. Further, followed by 1ns of equilibration, unrestrained MD simulation was performed for a period of 100ns in solvent. The Particle Mesh Ewald (PME) method was used to handle coulomb electrostatic interactions, while the LINear Constraint Solver (LINCS)s algorithmwas used to limit H-bonds. Using a cut-off value of 14 Å, the non-bonded contacts were trimmed. The trajectories generated were analyzed using some of the inbuild gromacs tools like ‘gmx rms,’ ‘gmx rmsf,’ ‘gmx hbond,’ ‘gmx gyrate,’ ‘gmx sasa,’ etc. and other additional packages for specific analysis wherever required. Conformational changes at the secondary structural level were monitored by using Dictionary of Protein Secondary Structure (DSSP) software (RRID:SCR_002725).

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Sterilization cycle perfomance are proven adequate during validation and it should be done in accordance to current international standards to guarantee that all critical parameters were met. Low temperature sterilization using vaporized hydrogen peroxide do not have a specific standard published yet and to validate the sterization cycles from this type of equipment, ISO 14937, the base standard for all sterilization cycle validation, should be followed. This standard uses biological indicator (BI) as an instrument for equipament performance evaluation and its cycle configuration must be according to half cycle approach. Since most of the manufactures do not have this option avaiable for the performance evaluation, cycles will canceled when the half cycle has been reached and the plasma phase concluded, when applicable. Since the US FDA is the only independent organism that has some type of product registration, equipments and self contained BIs that have been approved by them will be used in this study only.</p>

[BEFORE_START]
<p>Equipment must have been calibrated and passed thru preventive maintenance in the past month. Manufacturer must have concluded equipament instalation and operational qualification.</p>

[GUIDELINES]
<p>ISO 14937:2009 should be followed</p>

[STEPS]
?. 
?.
TITLE: Qiime 2 Workflow - Longitudinal study
AUTHORS: Carlotta Catozzi, Anna Cusco

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This workflow was performed using QIIME2 for a longitudinal study. The aim was to investigate the changes in mcirobiota after antibiotic and </span><span style = "font-style:italic;">Lactobacillus rhamnosus</span><span> treatment in water buffaloes affected by subclinical mastitis.</span></div></div>

[STEPS]
TITLE: Quality control analysis for 10X snRNA-seq 
AUTHORS: Daniel  Jacobsen, Dinh H Diep

[DESCRIPTION]
Here we describe a computational protocol for performing quality control analysis on shallow sequencing data obtained from 10X snRNA-seq experiments. The workflow starts with raw MiSeq run folders and uses cellranger to generate count matrices. The raw count matrices are analyzed and sequencing saturation plots are generated. The saturation plots are then compared against plots from a reference set of libraries with varying qualities (bad, fair, good, great), thus allowing for the determination of sequencing requirements as well as an assessment of the overall quality of each 10X snRNA experiment.

[STEPS]
1. Install cellranger using instructions from https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installation

 

2. Download the tar.gz file from this protocol.
3. Extract the tar.gz file from this protocol.
<FILENAME> is the name of the downloaded tar.gz file.
 

4. Install anaconda or miniconda Python distributions following given instructions.
Get anaconda from here: https://www.anaconda.com/products/distribution , OR
get miniconda from here: https://docs.conda.io/en/latest/miniconda.html
5. Install preseq using given instructions from http://smithlabresearch.org/software/preseq/.


 

9. Use conda to install bcl2fastq with the following terminal command:

 

10. Use conda to install required python packages with the following terminal command:

 

11. Run cellranger mkfastq to generate fastq files. Make sure that the following placeholders are set to the correct paths and desired names.
<FASTQ_OUT> is the name of the output folder
<RUN> is the path to the MiSeq run folder
<CSV> is the path to the sample-sheet.csv file

 

12. Run cellranger count. Make sure that the following placeholders are set to the correct paths and desired names.
<ID> is the name of the output folder for the sample
<SAMPLE> is the sample name used for the sample in the sample-sheet.csv file
<REF> is the path to the cellranger reference data folder
<NUM> is the number of expected cells from the experiment


 


13. Run the preseq script in the folder downloaded from this protocol. Make sure that the following placeholders are sset to the correct paths and names.

<PATH_TO_FOLDER> is the path to the folder that was extracted from the tar.gz file.
<ID> is the name of the output folder for the sample generated with cellranger.
 

14. View outputs.

<ID>.lc_extrap_log.txt contains preseq statistics
<ID>.lc_extrap_output.png to view the sequencing saturation plots
<ID>/outs/web_summary.html to view the cellranger analyses
<ID>/outs/summary.csv to view quality statistics generated by cellranger


6. Preseq requires the GSL libraries. Install GSL using the instructions from https://www.gnu.org/software/gsl/.

 
7. Create a symbolic link so that preseq can find the required gsl library.
 

8. Install samtools using given instructions from http://www.htslib.org/download/.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is from: <br /><span class="cit-title">Jian Cao, <em>et. al. </em>(2014) <a href="http://www.genetics.org/content/197/1/175.full" target="_blank">Insight into Insulin Secretion from Transcriptome and Genetic Analysis of Insulin-Producing Cells of Drosophila</a><br /></span>Genetics <span class="cit-vol">197<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">175</span><span class="cit-sep">-</span><span class="cit-last-page">192; </span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.113.160663<br /><br /></span>Please see the <a href="http://www.genetics.org/content/197/1/175.full" target="_blank">full manuscript</a> for additional details.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Mouse Whole Cell Tissue Processing for 10x Genomics Platform
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to isolate tissue from various ROI’s of the mouse brain followed by preparation of a highly concentrated single cell suspension for transcriptomic profiling on 10x Genomics platform.</div><div class = "text-block"><span style = "font-weight:bold;">Note:</span><span> 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.</span></div></div>

[STEPS]
TITLE: Immunostaining- Fluorescent
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Staining of mouse brain sections with immunofluroescent visualization.

[STEPS]
1. Wash freshly sectioned slices of tissue 2-3 times with 1X PBS to remove OCT. 
2. Block with 5% Normal donkey/Goat serum in 0.3% TRITON X-100 in 1X PBS for 60 min at Room temperature.

3. Wash sections 5 times in 1X PBS.

4. Following washes, transfer sections to a primary antibody solution containing a mix of primary antibody diluted in 0.3% TRITON X-100 in 1X PBS with 1% Normal Donkey/Goat serum 60 minat 4 °C.

5. The following day, wash sections 5 times in 1X PBS.
6. Transfer sections to secondary solution containing the adequate secondary antibody diluted in 0.3%
TRITON X-100 in 1X PBS with 1% Normal Donkey/Goat serum for 240 min atRoom temperature. 

7. Wash sections 3 times in 1X PBS.
8. Mount sections on microscope slides with medium (Vectashield with 4',6-diamidino-2-phenylindole (DAPI), H-1800-10).
TITLE: DAPI reconstitution and viability staining
AUTHORS: Emily  Stephenson

[DESCRIPTION]
For the reconstitution of DAPI to make a stock solution and dilution for a working solution.

Details of viability staining volumes also provided. 

[STEPS]
SECTION: Reconstitution
1. Add 2ml of water to the 10mg vial of DAPI to make a 14.3mM (5mg/mL) stock solution. Vortexing may be required if the powder does not dissolve with pipetting. 

(Note: DAPI will not dissolve in PBS)

Add a label with the date of reconstitution and store this stock solution at 4 degrees C for 6 months.
SECTION: Working Stock
2. Add 420uL DAPI stock to 20mL PBS to make a working solution of 300uM.

Store in the dark at 4 degrees C.
SECTION: For staining
3. For immunofluorescence, add to fixed cells at a concentration of 300nM (i.e. 1:1000 dilution for 5 mins then wash cells in PBS 3 times. 

For flow cytometry, add to live cells at a concentration of 3mM (i.e. 1:100)
TITLE: Detection of the rearrangement of exons 9-12 of the BRCA1 gene with the use of multiplex PCR
AUTHORS: Veronica Fragoso, Velazquez-Aragon JA, Alvarez-Gomez RM.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>In this protocol we developed a method for the detection of the rearrangement of exons 9-12 of </span><span style = "font-style:italic;">BRCA1</span><span> based on multiplex PCR. </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>A multiplex PCR is performed for the detection of the normal and mutated alleles of exons 9-12 of</span><span style = "font-style:italic;"> BRCA1</span><span> gene, 500 and 900 base pairs respectively, and a second PCR for the detection of the mutated allele. Both PCRs are observed by means of electrophoresis in 1.5% agarose gel.</span></div></div></div>

[STEPS]
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
Note: This mix is to one reaction, you must consider how many reactions will be done. Add ultrapure water to microtube of each mix (two mixes). Mix one  ; Mix two
14.1 µl
14.9 µl
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Add dNTPs 10Mm prepared with −N7-dGTP, 7-Deaza-dGTP
0.8 µl
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Add Betaine solution 5M
6 µl
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Add  the oligonucleotides en each one mix: Add to mix one (B01del9-12, B02del9-12 and B03del9-12) , ,,   respectively;  Add to mix two (B01del9-12, B03del9-12) ,
[10 pmol]
[10 pmol]
[10 pmol]
[10 pmol]
[10 pmol]
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Add the Taq DNA polymerase to the mix. Briefly vortex and centrifuge for some seconds.
[1.5 Units]
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Add and aliquot of DNA sample to each tube
[150 ng]
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Close perfectly the tubes and centrifuge for some seconds
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Put the reaction in the thermocycler
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
- Run the PCR following this conditions:  ;    -   10 cycles;  ,  ,  25 cycles; then  and the last
94 °C
94 °C
50 °C
72 °C
94 °C
55 °C
72 °C
72 °C
4 °C
?. [PCR mix to detection of the rearragement of exons 9-12 of BRCA1 gene]
Add 10x DreamTaq Buffer which includes MgCl2 at a concentration 20mM
3 µl
TITLE: Expression and purification protocol of GST-mCh-FYVE
AUTHORS: Liv  Jensen, Chunmei   Chang

[DESCRIPTION]
This protocol details the expression and purification of GST-mCherry-FYVE.

[STEPS]
SECTION: Protein expression
1. Transform the E.Coli BL21DE3 cells with plasmid encoding for GST-mCh-FYVE and plate them on Amp plate.
SECTION: Protein expression
2. Carry out protein expression in 1 L medium, induce with 200 micromolar (µM) IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at 18 °C  .

SECTION: Protein expression
3. Harvest the cells by spinning at 4500 x g for 20 min at 4 °C and stock at -80 °C until purification.

SECTION: Protein purification
4. Follow the GST batch purification by Size Exclusion Chromatography.
SECTION: Protein purification
5. Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at 16000 rpm at 4 °C for 60 min

SECTION: Protein purification
6. Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at 4 °C with gentle shaking for 30 min, apply to a gravity column, and wash extensively with Wash Buffer.

SECTION: Protein purification
7. Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer at 4 °C.

SECTION: Protein purification
8. Pool the peak fractions containing pure protein, snap-frozen in liquid nitrogen, and store at -80 °C.
TITLE: Squeeze” enrichment of intact cells (eukaryotic and prokaryotic) from marine sponge tissues prior to routine DNA extraction 
AUTHORS: Joe  Lopez

[DESCRIPTION]

A “Squeeze” method to enrich for intact cells (eukaryotic and prokaryotic) prior to DNA Extraction from marine sponge tissues

Most marine demosponge species possess a siliceous skeleton and spicules which can compose a high volume of the total biomass. This skeleton can be a priori removed before standard DNA extraction procedures (with CTAB, proteinase K, and various standard DNA extraction kit protocols).

The cell pellets derived from this protocol can be applied in other enrichment protocols prior to standard DNA extraction methods. For example, sponge cells may need to be separated from the large bulk of microbial symbionts by Percoll gradients. 

[STEPS]
2. With  sterile scalpel and forceps cut a piece of sponge mesohyl (tissue) approximately 1 - 2 cm3 place in a sterile petri dish. This can also be done on small fresh or frozen sponge pieces. If frozen, do not let sponge thaw completely but only enough to slice, since cell lysis may begin during thaw.
3. Soak sponge section by dripping 1- 3 ml "L buffer"  (10 mM Tris, ph. 7.6, 100 mM EDTA, 20 mM NaCl) on top of sponge tissue in the petri dish.  L buffer is derived from from the Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2001).The high EDTA concentration will protect DNA integrity.
1. Collect live or frozen sponge specimens, and have ready with all materials on the bench. 
4. Mince the tissue as small as possible with the sterile scalpel and forceps
5. Squeeze  or press the mesohyl pieces with forceps and against the petri dish bottom, expelling the contents of the cellular interior into the L buffer.
6. Collect the cells (~ 3ml) into microcentrifuge tubes
7. Centrifuge at top speed (10,000g) for 30 secs, or at half the speed if live cells are to be used downstream besides DNA extractions. 
8. Decant the supernatant;  SAVE THE PELLET– these are the cells both sponge and microbial that will be the source of DNA or other downstream procedures, such as cell fractionations via centrifugation.  The cell pellets can now be frozen for longer term storage.
9. Add an additional 1ml L buffer to rinse any remaining cells from the dish, mix with original tubes, and re-centrifuge
10. Decant supernatant, and save pellet
11. Spin 30sec more and remove remaining supernatant with a pipet; leave 10-20 ul of L buffer on pellet before frozen storage.
TITLE: Lesion Size 
AUTHORS: Cleo B.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Measuring the lesion size in </span><span style = "font-style:italic;">Brassica oleracea</span><span> after infection with </span><span style = "font-style:italic;">Xanthomonas campestris </span><span>pv. campestris. </span></div></div>

[STEPS]
?. Measure the lesion size on the leaf from the site of infection until the end of the yellow discoloration using a calliper.
TITLE: Vandy - Vertical Sleeve Gastrectomy in Mice
AUTHORS: Teri Stevenson, Vance L. Albaugh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This is the protocol for the vertical sleeve gastrectomy in the mouse. This procedure eliminates the greater curvature of the stomach leaving approximately 30% of the preoperative stomach capacity. Specifically, this procedure eliminates the greater curvature of the stomach where the ghrelin-producing cells are located. </div></div>

[STEPS]
?. Preoperative Care1. All animals must be singly-housed, given Ensure 12 hours before surgery and have all bedding removed. 2. Preoperative pain medications should be administered:     a. Ketoprofen (5 mg/kg)     b. Saline is given at the end of surgery and a second dose is given 24 hours later. 3. Ensure adequacy of anesthesia. 4. Place mouse on surgical board/surgical field over a warm water circulating blanket and immobilize gently. 5. Prep and drape animal sterilely.
?. Operation1. Begin the procedure by making a midline laparotomy incision with sharp surgical scissors. Be sure to stay on the linea alba and away from the rectus abdominus muscles. 2. Using cotton swabs, gently sweep the intestinal contents until the stomach is located. 3. Gently cut away all connective tissue around the stomach and ligate the vessel attached to the greater curvature of the stomach using cautery pen or 7-0 PDS II. 4. Place ligatures on all stomach vessels using 7-0 PDS II. 5. Using 6-0 monofilament suture a continuous line below your ligatures. 6. Remove greater curvature of the stomach by cutting below ligated vessels and monofilament line of suture. 7. Close stomach using 7-0 PDS II continuous pattern. 8. Next, close the abdomen in a simple, two-layer, interrupted fashion. The first interrupted layer should be a simple, interrupted muscle layer with 6-0 monocryl suture. Using good technique suture the connective tissue and not the muscle proper. The second layer is a skin layer also done in a simple, interrupted fashion with 7-0 Prolene suture.
?. Postoperative Care1. All mice receive 1.0 ml of warmed, sterile saline following the procedure before being placed in the recovery cages. 2. All mouse cages are kept partially on a veterinary-approved heating pad for 5 to 7 days postoperatively. 3. Mice will remain on an Ensure diet for 24 hours post-surgery with no bedding in the cage. 4. Mice are monitored until recovered from the procedure, which typically takes 7-14 days depending on the procedure. General behavior (i.e. bright/alert/responsive vs. depressed/obtunded) is monitored. 5. Pain medication is administered per protocol: ketoprofen 5 mg/kg post-op and again at 24 hours postoperative. 6. Additional pain medication may be needed depending on postoperative recovery.
TITLE: Small Drone Photos - Open Vegetation Protocol
AUTHORS: Sabine St-Jean, Etienne Laliberté

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here we describe the standardised protocol used by the </span><a href="http://www.caboscience.org/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Canadian Airborne Biodiversity Observatory</span></a><span> (CABO) to take small drone pictures of the plots and subplots in open vegetation surveys. In 2019, such pictures were taken at the Mer Bleue Bog (Ontario) and Parc national des Îles-de-Boucherville (Québec) sites. These pictures are to be classified later (see </span><a href="https://www.protocols.io/view/post-processing-abundance-and-distribution-of-spec-9mrh456" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">the Post Processing protocol</span></a><span>) to obtain the abundance and distribution of plant species through every subplot.</span></div></div>

[STEPS]
?. [Picture-Taking]
Fly the small drone to take photos of the plots and subplots.
?. [Photo Import]
Import the photos.
?. [Picture-Taking]
Turn on the drone and the controller. Connect the controller to your phone and open the DJI Go 4 App. Press Go Fly. Adjust the settings to Speed = 1:1000 and Iso = 400. If it is very sunny, set the Iso to a lower value; if darker, to a higher value. Activate the Grid option to view lines of the center of the image.
?. [Picture-Taking]
Take off.
Before taking each picture, make sure:the camera is at a 90° angle (facing down completely), the plot or subplot is centered and its sides are visible, and the drone is still.
?. [Picture-Taking]
Take a photo of the whole plot, with the southwest corner of the grid being in the lower-left corner of the picture.
?. [Picture-Taking]
Take the photos of each subplot, in the same order as the subplots numbers.
?. [Picture-Taking]
Land the drone on the platform.
?. [Picture-Taking]
From the DJI Go 4 App → Editor → Album → Photo, download all of the original pictures to your cellphone. Turn off the drone and the controller and put them back in their case until the next flight.
?. [Photo Import]
From your cellphone Photo App → DJI album, move the 10 pictures from the plot (1 for the plot and 9 for the subplots) to a new album titled with the plot name.
?. [Photo Import]
Crop each picture to limit the area outside the plot or subplots' perimeter. The edges of the PVC pipes forming the plot or subplot of interest should remain visible.
Once the photos of a given plot have been put in a specific album (step 2.1), steps 2.2 to 2.4 can be done later (i.e., outside the field) to maximize efficiency on the field.
?. [Photo Import]
Upload the picture of the plot to Fulcrum, under Vegetation Surveys: Herbs and Shrubs → Survey Event → Vegetation Photos: Plot.
?. [Photo Import]
Upload the picture of the subplots to Vegetation Surveys: Herbs and Shrubs → Cover Estimates: Subplot → Subplots → Record item [1 to 9] → Vegetation Photos: Subplot.