eamag/protocols-with-steps huggingface dataset:
TITLE: Pilot Exercise: Generating the Illumina SampleSheet and sharing data via BaseSpace
AUTHORS: Ruth  Timme, Maria   Balkey, Tunc Kayikcioglu, Candace Bias, Cameron Boerner, James Pettengill

[DESCRIPTION]
This protocol describes the required steps to generate Illumina SampleSheet and provides guidelines for sharing data via BaseSpace with FDA for the 2021 Pilot Exercise 

[STEPS]
SECTION: Generating Sequencing SampleSheet
1. Create your SampleSheet using Excel or a text editor.  Name your sample sheet according your internal protocols, and use a *.csv extension.    The sample sheet is organized in sections titled Header, Reads, Settings and Data.  Section headings are case-sensitive and shown in brackets [ ].  Details for each section are described below. 

SECTION: Generating Sequencing SampleSheet
1.1. Header Section. 


 

SECTION: Generating Sequencing SampleSheet
1.4. Data Section:  Include the machine ID in the Sample_Name column.  e.g. sample_B01_M01234 where M01234 corresponds to the machine ID. This is the ID that will get populated to the fastq file names.

         Sample_IDSample_NameSample_PlateSample_WellI7_Index_IDindexI5_Index_IDindex2Sample_ProjectDescriptionWPP-sample_B01sample_B01_M12341A1SU001TTGTTCCTTGI5_1CCGAACAACAPilotWPP-sample_C01sample_C01_M12341B1SU002AGGTTGTGTTI5_2AAGAATCGGCPilotWPP-sample_SA_101sample_SA_101_M12341C1SU003TATTGGTTGGI5_3CACTTCGCTTPilotWPP-sample_SA_201sample_SA_201_M12341D1SU004CACTTCGCTTI5_4TATTGGTTGGPilot
 
SECTION: Generating Sequencing SampleSheet
1.5. If a new library or sequencing protocol is utilized for processing the pilot exercise samples, make the changes in the corresponding parameters for the sequencing sample sheet.  
SECTION: Generating Sequencing SampleSheet
1.2. Reads Section

**Enter the length of reads you are generating (could be different from 151)

 

SECTION: Generating Sequencing SampleSheet
1.3. Settings Section


 

SECTION: Transfer sequencing data through BaseSpace
3. Click the "Share Project" option to share the run using an email address. 

SECTION: Transfer sequencing data through BaseSpace
3.1. Click the Runs tab.
SECTION: Transfer sequencing data through BaseSpace
3.2. Select the run that you would like to share with the FDA LFFM wastewater project team.
SECTION: Transfer sequencing data through BaseSpace
3.3. Click Share.
SECTION: Transfer sequencing data through BaseSpace
3.4. Enter the email address for the FDA team (covidtrakr@fda.hhs.gov), and then click Add Collaborator.
SECTION: Transfer sequencing data through BaseSpace
3.5. Click Save Settings. Your run will be automatically shared with the FDA LFFM wastewater project team.

  



SECTION: Generating Sequencing SampleSheet
2. Starting your MiSeq run and connecting to BaseSpace:

When you set up the run on the MiSeq, select the option to log in to BaseSpace.
TITLE: Isolation of natural killer (NK) cells from human blood products
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
Standard isolation procedure for peripheral blood mononuclear cells from human blood leukopheresis products

[STEPS]
1. Healthy donor blood products (Leukopaks) were obtained from Memorial Blood Bank (Minneapolis, MN). All samples were de-identified and their use was approved by the University of Minnesota and NMDP institutional review board in accordance with the Declaration of Helsinki.
2. Peripheral blood mononuclear cells (PBMCs) were separated through density gradient filtratation (Ficoll-Paque; GE Healthcare). 
3. For experiments with enriched NK cells, PBMCs were processed fresh using negative magnetic bead separation (EasySep Human NK Cell Enrichment Kit; Cat. No:19055, STEMCELL Technologies). 
4. Experiments with PBMCs or enriched NK cells were performed in RPMI 1640 supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. Where specified, culture was supplemented with recombinant human IL-15 (National Cancer Institute).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The spinal bones of knockout mice were fixed in ethanol, stained by the Villanueva method, and embedded in methyl methacrylate without decalcification. The administration schedule involved a single subcutaneous injection of tetracycline administered to null and wild-type mice, followed by no administration for the next two days. Thereafter, they were given a single subcutaneous injection of calcein, followed by no administration on the next day. The results of the bone histomorphometric analysis are expressed according to the methods of the ASBMR Histomorphometry Nomenclature Committee, excluding the osteocyte parameters.</p>

[STEPS]
?.
TITLE: Methods for Visualization of Pig Vagus Nerve &#34;Vagotopy&#34; Using Ultrasound
AUTHORS: Megan  Settell, Rex  Chen, Kip  Ludwig

[DESCRIPTION]
This protocol describes the surgical, ultrasound, and histology methods for visualizing pig vagus nerve vagotopy.

[STEPS]
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
1. Surgically Expose the Vagus Nerve
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
2. Place wire under the vagus nerve every 2-3 cm, where histology samples will be taken.
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
1.1. Place subject in dorsal recumbence position.

SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
1.2. Expose the length of the cervical vagus nerve by creating an incision (15-20 cm long) approximately 3 cm lateral and parallel to midline starting at the level of the mandible.
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
1.3. Divide tissue using blunt dissection to expose the carotid sheath, identifying and removing connective tissue from the carotid artery, vagus nerve and jugular vein.
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
1.4. Particular attention should be paid to clearing the vagus nerve at the most cranial point of interest (superior cervical ganglion, nodose/inferior cervical ganglion) to the caudal region of interest.
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
2.1. Depending on the diameter and type of wire, artifact will be created in the ultrasound to allow for histological matching.  (ex: Silver (Ag) 0.015'' bare wire)
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
3. Fill the surgical pocket with room temperature/heated 0.9% Sodium Chloride (saline).
SECTION: Histology of Vagus Nerve Following Ultrasound
5. Pat the vagus nerve dry with surgical gauze, or cotton tipped swabs above each wire.
SECTION: Histology of Vagus Nerve Following Ultrasound
6. Place histology dye as desired to mark the location of each wire along the length of the vagus nerve.
SECTION: Histology of Vagus Nerve Following Ultrasound
6.1. Recommended Histology Dye: Bradley Products, Inc., Davidson Marking System, Bloomington, MN.
SECTION: Histology of Vagus Nerve Following Ultrasound
7. Once histology dye has been placed in the desired locations, remove the vagus nerve and place in 10% Neutral Buffered Formalin for histological processing.
SECTION: Histology of Vagus Nerve Following Ultrasound
8. For Gomori's trichrome staining, please see our previously published protocol: dx.doi.org/10.17504/protocols.io.9ieh4be
SECTION: Preparing Surgical Pocket for Vagus Nerve Ultrasound
4. Collect ultrasound in regions of interest, or along the length of the vagus nerve.
TITLE: 1: User-friendly protocol: Probe set design (SABER-FISH)
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol offers step by step instructions for probe design for given organism (standard mRNA detection).</div></div>

[STEPS]
?. Retrieve BED file for gene or gene region of interset from UCSC genome browser:50
Genome-wide probe sets have already been generated for a variety of stringency levels. The probe set files with medium probes end in ‘b’, for instance the ‘mm10 chr16b.bed’ file. B stands for ‘balanced.’ Probe sets by chromosome for a variety of organisms are available on The Wu lab Oligopaints website: https://oligopaints.hms.harvard.edu/.
Furthermore, a new online tool for automated FISH probe design is available at http://paintshop.io/ (https://www.biorxiv.org/content/10.1101/2020.07.05.188797v1 ).
?. Select appropriate genome (e.g. mm10 for mouse).
?. Bring gene (or region) of interest into field of view.
?. tools → table browser.
?. Fill in as follows:Group: genes and gene predictionsTrack: e.g. NCBI RefSeqTable: e.g. RefSeqRegion: PositionOutput format: BED
?. Get output → exons (for exon FISH).
?. Check file.
?. If there are multiple isoforms (e.g. different names that start with NM), then manually delete all but the desired one.
?. Check the strand orientation. If (-), then no need for extra steps. If (+), then you must get the reverse complement after probe design.
?. Example UNIX commands for identifying overlapping probes:Ensure Biopython and BEDTools are installed and loaded (see above), or transfer BED file and probeset files to a cluster/machine that has these set upA symbolic link to the chromosome probe set file can be placed in the working directory using: ln -s ../oligopaints/mm10_chrNb.bedFor the gene of interest (GOI) bed file, run intersectbed (bedtools): intersectBed -a mm10 chrNb.bed -b GOI.bed -f 1 > GOI_probes.bedIf the gene is the (+) strand, take the reverse complement: python ../bin/probeRC.py -f GOI_probes.bed -o GOI_probesRC
?. Primer sequences are appended to the 3’ end of the primers with a linker of TTT, e.g.: (probe sequence)TTT(9-mer primer sequence)
The optimal number of required probes for each mRNA target varies based on considerations such as which fluor will be used for detection and whether branching will be employed as well as transcript length and sequence (e.g. homology to the genome). In tissues, we successfully detected RNAs across a variety of fluors using anywhere from 24 to 50 probes. We recommend starting with this range of probe set sizes.
TITLE: BAF_Protocol_010 Metabolomics LC-MS(/MS): Vanquish UPLC and Orbitrap ID-X
AUTHORS: Nicholas Sherman

[DESCRIPTION]
This protocol is the basic LC and MS running parameters for metabolite runs. The samples typically have fewer components (ions) than proteomics so the gradients are shorter and most molecules are singly charged. 

[GUIDELINES]
These are our general settings as a starting point for untargeted metabolomics experiments. Specific adjustments need to be made to meet specific samples or type of data required.

[STEPS]
SECTION: Prepare samples for injection
1. Suspend dried metabolite extracted samples with 100 µL  of 0.1% Formic acid in water containing 100X diluted Metabolomics QRESS heavy labeled standards. 

SECTION: Prepare samples for injection
2. Vortex, microfuge for 15 min  at max speed, and take off 50 µL into autosampler vial (do not touch bottom of tube). 

SECTION: Prepare samples for injection
3. Make sure there is no air in the bottom of the vial, carefully add the vial into the autosampler of the UPLC Vanquish system.
SECTION: Prepare samples for injection
4. In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 20% of sample.
SECTION: LC parameters 15 min gradient
5. Speed limits: draw speed 1 uL/s; dispense speed 1 uL/s.
Injection wash parameters: Wash mode: both; Wash time: 10.0s; Wash speed: 10.0 uL/s.
Column temperature: 30 °C 
Run time: 15 min
Solvents: %A1: Water, 0.1%FA. 
                %A2: Methanol, 0.1% FA.
Upper limit pressure: 1500 bar
Flow = 0.250 ml/min
Equilibration: -3min, 0% B, curve = 5.
Gradient (all steps curve = 5): 0 min 5%B; 8 min
50% B; 8-9 min 98% B; 9-13 98% B;  13-13.1
min 0%B; 13.1-15 min 95%B.
SECTION: General Instrument (Orbitrap ID-X) – Positive and Negative modes
6. Ion Source Type: H-ESI
Spray Voltage Positive 3500V 
Spray Voltage Negative 2500V
Ion Transfer Tube Temperature 275 °C, Sheath gas 35, Aux Gas 7, Sweep gas 2, Vaporizer Temp 320 °C
Default charge state: 1 
SECTION: Full scan parameters
7. Scan 67-1000 m/z, resolution 60K, Quadrupole isolation: True, normalized AGC 25%, RF lens 35%, Max. Inj. Time 50ms, 1 microscan, Data Type Profile, Polarity Positive or Negative (create two methods one for Positive acquistion and another for Negative acquisition)
SECTION: MS/MS parameters
8. Min intensity 2E4, Dynamic exclusion - 1 time, 2.5s, 10ppm, isolation 1.5 m/z, resolution 15K, HCD NCE stepped 20, 35, 50%, Max. Inj. Time 25ms, normalized AGC 25%, 1 microscan, Data Type Centroid.
TITLE: SARS-CoV-2 RBD ELISA
AUTHORS: Anita Iyer

[STEPS]
?. Coating:-Dilute SARS-CoV-2 RBD antigen to 1 µg/mL in carbonate buffer (20 mL per 384 well plate).-Add 50 µl/well of this antigen to a 384 well plate (200µl/ well for 96 well plate).-Cover with plate seal. Using a microcentrifuge, quick spin plates to allow the solution to settle at the bottom.-Incubate at room temperature for 1 hour.-Wash 3x (300 µl/well for 96 well plate, 100µl/well for 384 well plate) in high salt TBST and blot plate dry on paper towels.
?. Before you start:-Refer to the materials and recipes prior to starting the experiment.-Critical that carbonate buffer, sample diluent, substrate, and substrate buffer be at room temperature before use.-Make blocking and sample buffer 30 mins before assay to allow solubilization of milk powder.
?. Blocking:-Add 5% Milk-1X TBS (96 well plate: 300 µl/well; 384 well plate: 100 µl/well) to the assay plate. -Cover with plate seal. Using a microcentrifuge, quick spin plates to allow the solution to settle at the bottom.-Incubate at room temperature for 30 minutes.-During the blocking incubation, prepare samples and standards by diluting in 5% Milk- 1X TBST as appropriate or indicated below.-At the end of 30 mins, wash the plate 3x in high salt TBST (300 µl/well for 96 well plate, 100µl/well for 384 well plate) and blot plate dry on paper towels.
?. Samples and Standard Preparation and Addition:-Serially dilute samples 1:100, 1:400, 1:1600 and 1:6400 in 5% Milk- 1X TBST.-For monoclonal standards, perform a 2-fold, 8 series dilution starting at 0.025 µg/ml for IgG and 0.25 µg/ml for IgA and IgM. Transfer standards to 96 well round-bottom plate. -At the end of blocking step, transfer samples and standards (96 well plate: 100 ul/well; 384 well plate: 25 ul/well) to the assay plate that was coated with RBD and blocked in steps above. -Cover with plate seal. Using a microcentrifuge, quick spin plates to allow the solution to settle at the bottom.-Incubate for 1 hour at 37°C on a plate shaker set to 100 rpm.At the end of 1-hour incubation, wash 5x in high salt TBST (300 µl/well for 96 well plate, 100µl/well for 384 well plate) and blot plate dry on paper towels.
?. Secondary:-Add secondary at the following dilutions in 5% Milk-1X TBST (96 well plate: 100 ul/well; 384 well plate: 25 ul/well)a.IgG 1:10,000b.IgA 1:5,000c.IgM 1:10,000-Cover with plate seal. Using a microcentrifuge, quick spin plates to allow the solution to settle at the bottom.-Incubate for 30 minutes at room temp on a plate shaker set to 100 rpm.-Wash 5x in high salt TBST and once with 1X TBS (300 µl/well for 96 well plate, 100µl/well for 384 well plate) and blot plate dry on paper towels.
?. Development-Make sure TMB equilibrated to room temp 30 mins before development.-Add appropriate volume of TMB to wells (96 well plate: 100 ul/well; 384 well plate: 25 ul/well). Start a timer as soon as you add substrate  to the first set of wells. -Cover with plate seal and spin down using a microcentrifuge. Incubate in dark for 5 minutes. -Add 2N stop H2SO4stop solution (96 well plate: 100 ul/well; 384 well plate: 25 ul/well)-Read plates at 450 nM – 570 nm.
TITLE: 100ml EB buffer (PH=8)
AUTHORS: Menglin WANG

[DESCRIPTION]
This is the recipe for how to make 100ml EB buffer (PH=8)

[STEPS]
SECTION: 100ml EB buffer (10mM Tris-HCl)  pH=8
1. Prepare 80 mL    in a suitable container. 

SECTION: 100ml EB buffer (10mM Tris-HCl)  pH=8
2.  Add 0.12114 g of    to the solution.

SECTION: 100ml EB buffer (10mM Tris-HCl)  pH=8
3. Adjust solution to desired (pH 8.0) using HCl (typically pH ≈ 7.0).
SECTION: 100ml EB buffer (10mM Tris-HCl)  pH=8
4. Add   until the volume reaches to 100ml.
TITLE: Passaging of cells
AUTHORS: Guido  Krähenbühl

[DESCRIPTION]
General Lab procedure, Cell passage


[BEFORE_START]
Warm up culture media, PBS and Trypsin in a 37 °C 


[STEPS]
1. Inspect cells for confluency, when 70-90 % confluency is reached cells are ready for passage
2. Discard media and wash once with PBS
3. Add Trypsin to fully cover the surface of the culture vessel
4. Incubate for 5 min at 37 °C 

4.1. Lightly tap the culture vessel and check for cell detachement. If cells are still attached prolong incubation
5. Add 2/3 of culture media to 1/3 of trypsin and transfer cells into a Falcon tube

6. Centrifuge at 300 rcf, 5 min 

7. Discard supernatant and resubstitute with 1 mL 

8. Count cells and adjust media volume for required cell density
9. Plate cells
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For <a href="https://www.protocols.io/view/Transcriptomics-During-One-Step-Growth-Curves-for-dem3c5" target="_blank">Transcriptomics During One-Step Growth Curves for Cellulophaga Phages protocol</a>.

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

[DESCRIPTION]
<p>Basic protocol to achieve lipofectamine transfection of Human Embryonic Kidney 293 (HEK293) cells with cDNA and/or siRNA.</p>

[BEFORE_START]
<p style="text-align: left;">Make complete DMEM:</p>
<p style="text-align: center;"> </p>
<table style="height: 105px;" width="312">
<tbody>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;"><strong><em>Reagent</em></strong></td>
<td style="width: 148px;"><strong><em>Volume</em></strong></td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">DMEM</td>
<td style="width: 148px; text-align: center;">432.5 mL</td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">FBS</td>
<td style="width: 148px; text-align: center;">50 mL</td>
</tr>
<tr style="text-align: center;">
<td style="width: 148px; text-align: center;">Pen/Strep</td>
<td style="width: 148px; text-align: center;">5 mL</td>
</tr>
<tr>
<td style="width: 148px; text-align: center;">HEPES (1M, pH 7.4)</td>
<td style="width: 148px; text-align: center;">12.5 mL</td>
</tr>
</tbody>
</table>
<p> </p>

[GUIDELINES]
<p>If protein yields are low following this method, it may be worthwhile to use complete DMEM without Pen/Strep. In this protocol, we op to use complete DMEM with Pen/Strep even though yields may be lower.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: genomic DNA-extraction 
AUTHORS: Michael   Burgis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">DNA isolation protocol for quick and rapid extraction of genomic DNA from Chlamydomonas reinhardtii cells. (also works for cyanobacteria)</div><div class = "text-block"><span>This protocol is based on a paper published by Newman et al 1990 (PMID: </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/1981764" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">1981764</span></a><span>).</span></div><div class = "text-block">The protocol is designed to be used for single colonies or 1ml of cell culture but can be used for a larger amount of biomass if scaled correctly. Here I describe the protocol for 10ml of overnight culture.</div></div>

[STEPS]
?. [Buffer preparation]
TEN Buffer (10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl)SDS-EB Buffer (2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0)TE Buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0) [autoclaved]
?. [Resuspension and lysis]
Pellet the 10ml overnight culture of your algae or cyanobacteria in a 15ml falcon at 5.000g.
?. [Resuspension and lysis]
Resuspend in 5ml of TEN Buffer by vortexing, spin for 10 sec. and aspirate off supernatant.
?. [Resuspension and lysis]
Resuspend cells in 150 ul H2O on ice and add 300 ul of SDS-EB buffer, vortex to mix.
?. [Phenol Chloroform washing]
Extract once with 3.5 ml phenol/Chloroform:isoamyl alcohol(1:1) for a few min by vortexing, separate phases by centrifugation for 5 min., transfer aq.  phase to a new falcon tube.
?. [Phenol Chloroform washing]
Extract once with 3 ml Chloroform:isoamyl alcohol (24:1), transfer aq. to a new tube.
?. [Phenol Chloroform washing]
Repeat step in order to maximize purity.
?. [Precipitation and Resuspension]
Add 2 volumes abs. ethanol, incubate on ice for 30 min., centrifuge for 10 min., wash pellet once with 200 ul 70% ethanol.
?. [Precipitation and Resuspension]
Dry at 60°C until all ethanol evaporated and resuspend in desired volume of TE Buffer.
?. [Precipitation and Resuspension]
Incubate at 50 °C for 1 to 2 h to ensure complete re-suspension. (Can be extended to overnight incubation)
?. [Precipitation and Resuspension]
Measure the quality and quantity of your DNA using a NanoDrop.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Recent attempts to replicate the ego depletion effect have often failed. The goal of this study is to shed light on research experiences and practices regarding ego depletion. A PubMed search resulted in 1721 researchers who had previously published on ego depletion. They were invited to participate in an anonymous online survey. The respondents (<em>n</em> = 277), on average, had published over three papers on ego depletion, and had completed more than two additional, unpublished studies. Respondents indicated that in more than 40% of their studies, results were similar in magnitude to those reported in the existing literature, and more than 60% reported conducting a priori power analyses. 39.2% of respondents were aware of other researchers who engaged in questionable research practices, while 37.7% affirmed to have used questionable research practices. These results underline the importance of reducing questionable research practices to reliably test the validity of the strength model.</p>

[STEPS]
TITLE: Seawater sample preparation for microplastic determination
AUTHORS: Mª Dolores Gelado Caballero, Tania Montoto Martínez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol is fit for seawater sample preparation prior to its observation under a stereomicroscope for the determination or microplastic particles. </div></div>

[STEPS]
?. Clean the glass beaker where your seawater sample is transfered: rinse it thoroughly three times withNote down the volume.
?. Prepare the  vacuum filtration system with a glass microfibre filter (0.7 μm; GF/F ø=47 mm) and filter the full volume.
?. Rinse the vacuum filtration system three times while it continues filtrating to the microfibre filter, so no particles are left behind.
?. Place the filter on a  petri dish and let it dry overnight in the oven at  .
60 °C
?. Observe the filter under a microscope. Take pictures and measures of the identified particles following Lusher et al (2014) guidelines.