eamag/protocols-with-steps huggingface dataset:
TITLE: Canine Respiratory Pathogen Detection Assays
AUTHORS: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

[DESCRIPTION]
The Canine Respiratory Pathogen (CRP) Detection Assays is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of canine adenovirus-2 (CAdV-2), canine distemper (CDV), canine herpesvirus type 1 (CHV-1), canine influenza A virus (CIV), canine parainfluenza (CPiV), canine pneumovirus (CPnV), canine respiratory coronavirus (CRCoV), SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos and Mycoplasma canis in nasal and pharyngeal swab samples.

[GUIDELINES]
Shipping and Storage 
The CRP Detection Assays are shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at  -80°C.

Limitation:
Strict compliance with the instructions is required for optimal results.
Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
The presence of RT-PCR inhibitors may cause false negatives.
Results of Canine Influenza A Subtypes Identification Assay need to be interpreted in consideration of all clinical and laboratory findings.

Quality Control:
The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA or plasmid copies number. All assays have a limit of detection (LOD95)  60 copies/l.


[STEPS]
SECTION: Reaction Setup
1. Thaw all reagents on ice.
SECTION: Reaction Setup
2. Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice 
 

SECTION: Programming the Thermocycler
4. The following fluorescence channels should be selected: ABYTM, FAMTM, JUNTM, and VICTM.

SECTION: Programming the Thermocycler
5. ROXTM should be used as a passive reference dye.
SECTION: Programming the Thermocycler
6. The standard mode should be selected. Setup cycling condition following table 2: 



 

 

SECTION: Results interpretation
7. Before results analysis, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.
SECTION: Results interpretation
8. Each assay is considered valid if the following criteria are met:
  
SECTION: Results interpretation
9. The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
   
 

SECTION: Reaction Setup
3. Setup the Master Mix according to the following table 1:
TITLE: Cylinder behavioral test (mice version)
AUTHORS: miquel.vila

[DESCRIPTION]
Cylinder behavioral test for left and right forepaw use in mice

[BEFORE_START]
All behavioral tests have to be performed during the light cycle by an investigator blinded to the experimental groups.

[GUIDELINES]
Important: Rats that present an asymmetric usage of the right-left forepaws at basal examination need to be excluded from the experiment. 

[STEPS]
1. First allow mice to habituate to the experimental room for at least 1 h before each test
2. Put animals individually in a glass cylinder and the total number of left and right forepaw touches performed within 5 min was counted
3. Clean behavioral equipment with 70% ethanol after each animal has been tested to avoid olfactory cues
TITLE: Sandwich ELISA for investigating the binding of Protein-LG (SpLG) to avian immunoglobulins  using  anti-IgY-peroxidase as conjugate.
AUTHORS: Angel  Justiz-Vaillant, Monica F. Smikle

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

[STEPS]
?. This ELISA is used to study the interaction of protein-LG (SpLG) with diverse avian immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of recombinant SpLG or a mixture of SpL with SpG in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. 50 µl of  avian egg yolk or egg white (1 mg/ml) is added and incubated for 1.30h at RT and the microplate is then rewashed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-anti-IgY conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point can be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the binding affinity of SpLG to avian immunoglobulins.
TITLE: Study Design (Part 3 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 3 of  "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: Bleaching a population of C. elegans nematode worm in a tube
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the C. elegans population bleaching protocol.</div></div>

[STEPS]
?. 1. Use C. elegans plates that have many gravid hermaphrodites (only the eggs will resist to the bleach treatment). Wash the plates with M9 solution: pipet M9 solution across the plate several times to loosen worms and eggs that are stuck in the bacteria. Collect the M9+worms solution in a 15 ml Falcon tube. Top up the volume to 14 ml M9. Tip: Adult hermaphrodites have a tendency to stick to the walls of plastic pipettes. To avoid this issue when harvesting the worms, you can: (i) add a small amount of detergent (NP40 0.005% or tween) to the M9 used to harvest the worms; or (ii) pre-wet the plastic pipette in M9+detergent; or (iii) use glass pipettes.Tip: Pipette gently to avoid collecting bits of agar together with the worms.
?. 2.  Wash the animals three times (or until the worm suspension is clear of bacteria) with 14 ml M9. To do so, centrifuge the animals at 2,000 rpm for 2 minutes, discard the supernatant with the vacuum pump, re-suspend the worm pellet in 14 ml M9. After the last wash, do not re-suspend the worm pellet in M9 and proceed to step 3.
?. 3. Add 5 mL of bleaching solution to the worm pellet and immediately proceed to the next step.Optional: depending on the number for worms/level of contamination, you can increase the volume of bleach solution from 5 ml to 6 ml. You can also use stronger bleaching solution (3 mL NaOH 10M, 7.5 mL Bleach, 40mL H2O). The standard conditions should work most of the time, however.
?. 4. Vortex the tube for 6 minutes. Then quickly check the mixture under a dissecting microscope (through the flacon tube). The worms should be dissolved and only eggs should be visible. If so, immediately proceed to the next step.Note: If necessary, you can vortex 2 additional minutes. Do not over bleach though! If the eggs stay too long in the bleach solution, they will die
?. 5. Add some sterile M9 to the mixture of dissolved worms/eggs in bleaching solution (up to 14 ml final volume). Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump and immediately proceed to the next step.
?. 6. Resuspend the egg pellet in 14mL sterile M9. Vortex the mixture for five seconds, holding the tube horizontally. Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump. Repeat this step three times.
?. 7. After the last wash, resuspend the egg pellet in the desired volume. You can either pipette the eggs onto an NGM plate (use a glass pipette) or leave the eggs in liquid suspension (4 ml of sterile M9 recommended) and place them on the rotating wheel (20°C room) overnight (you will have synchronised L1 on the next day).
TITLE: Generation of pLKO.1 Plasmids Containing shRNA
AUTHORS: Shiyi Wang

[DESCRIPTION]
Generation of pLKO.1 Plasmids Containing shRNA

[STEPS]
1. **Obtain shRNA Plasmids**
1.1. - Obtain pLKO.1 Puro plasmids containing shRNA sequences against mouse/rat Lrrk2 (shLrrk2: TRCN0000322193) and mouse/rat Atg7 (shAtg7: TRCN0000092163) from the RNAi Consortium (TRC) via Dharmacon.
2. **Synthesize Scrambled shRNA Sequences**
2.1. - Generate two scrambled shRNA sequences: GTTGCTGAATGGCGGATCTAT and GTTGCGGTTATGAATAGTACT.
3. **Cloning into pLKO.1 Vector**
3.1. - Clone the scrambled shRNA sequences into the pLKO.1 TRC cloning vector254 following the protocols provided by Addgene (https://www.addgene.org/protocols/plko/).
4. **pLKO.1-shRNA-EGFP Plasmid Construction**
4.1. - Remove CAG-EGFP from pLenLox-shNL1-CAG-EGFP255.
4.2. - Insert the CAG-EGFP sequence between the KpnI and SpeI sites in the pLKO.1 Puro vector, replacing the puromycin resistance gene to generate pLKO.1-shRNA-EGFP.
5. **pLKO.1-shRNA-mCherry Plasmid Construction**
5.1. - Replace EGFP with mCherry between the KpnI and NheI sites in the pLKO.1 Puro vector to generate pLKO.1-shRNA-mCherry plasmids.
6. Notes:
6.1. - Validate the integrity and orientation of cloned sequences through restriction digestion and sequencing.
6.2. - Use sterile techniques and appropriate safety measures when working with plasmid DNA.
TITLE: WIPI2d puncta formation assay
AUTHORS: Dan Tudorica

[DESCRIPTION]
Labels sites of autophagosome generation.

[STEPS]
1. Target cells are cultured until they reach 80% confluence on cell-culture treated, chambered confocal slide, and then transfect cells with a WIPI2d-Halo mammalian expression plasmid using lipofectamine 3000 (Thermo Fisher). Calculate DNA amount to use via the manufacturer instructions, scale DNA according to culture area.
2. Wash cells into antibiotic free medium prior to transfecting. Incubate the cells for 24 H post transfection, then wash cells into fresh medium. Incubate for an additional 24 H before imaging.
3. On day of experiment, replace medium with growth medium supplemented with 10 uM Oligomycin A, 5 uM Antimycin A (OA). Incubate for 45 min.
4. Add 1x cell permeable, fluorescent Halo Ligand diluted in OA medium to label. Incubate for 15 min before washing cells twice with PBS and fixing in 4% PFA. Wash once more with PBS. Image cells immediately on a confocal microscope.
5. To analyze open images in FIJI, find a cell with largely cytosolic WIPI signal, and measure. Subtract this value from the image as a whole using the math > subtract function. 
6. Define ROIs around target cells and measure total signal using FIJI's native measure function. Inspect cells to make sure cytosolic WIPI signal is being reliably eliminated, and most of the fluorescence intensity comes from punctate WIPI.
TITLE: RNAlater preparation 
AUTHORS: Tsu-Chun Hung

[DESCRIPTION]
RNAlater preparation

[STEPS]
SECTION: DEPC-treated water prepartion
1. Prepare 0.1% DEPC-treated water and autocleave it
 

SECTION: RNAlater preparation
2. Add these component in 1L bottle in order
 

SECTION: RNAlater preparation
3. Put the bottle on the stirring hot plate, stir till the mixture clear

 


SECTION: RNAlater preparation
4. After cool down, add 8.96 mL 1M sulfuric acid to adjust pH to 5.2
TITLE: MRI Imaging of the Gut 
AUTHORS: Santiago Unda, rar, Michael G. Kaplitt

[DESCRIPTION]
This test is used measure peristaltic activity in the upper portion of the gut. 


[STEPS]
6. Localize the longitudinal axis of the stomach.
SECTION: Protocol
1. Habituate mice to eat Diet gel (Cat#DietGel Recovery, ClearH2O, ME, USA) for at least a week. 
 

SECTION: Protocol
2. Fast mice for 12 to 18 hours.
SECTION: Protocol
3. Prepare 10g of Diet gel mixed with 22.4mg of Gadolinium [Gd-DTPA powder] (Cat#381667, Sigma Aldrich, St. Louis, USA). 
 

SECTION: Protocol
4. Give 0.3g of Diet Gel-Gadolinium mix to each mouse. 
 

 

5. Using a 7T rodent MRI (BioSpec 70/30; Bruker Instruments, Billerica, USA), position the animal in prone and monitor respiratory activity patterns. 
 

SECTION: Notes
11.       Localize the longitudinal axis of the stomach. 
7. Perform series of alternating volumetric and fast scans. 
 

8. Sequences commonly take 2 min, repeat for as long as your experimental setting requires.
TITLE: Ln-DAB2 Solutions
AUTHORS: Stephen R. Adams, Mason R. Mackey, Roger Tsien, Mark Ellisman, Jeffrey D. Martell

[DESCRIPTION]
We use multicolor EM (electron microscopy) to paint multiple cellular markers by locally depositing specific Ln3+ from  prepared solutions of Ln-DAB2 by mSOG, APEX2 or HRP. Each Ln3+ is then visualized by electron energy-loss spectroscopy and energy-filtered EM. Elemental maps are overlaid on conventional EM give multicolor EM. 

[GUIDELINES]
Ln-DAB2 solutions in cacodylate buffer were prepared immediately before use at room temperature. 

Note: This buffer is hazardous and any waste should be handled accordingly. 

[STEPS]
1. To make 10 mL of a 2 mM Ln, Ce or Pr-DAB2 solution, 15.6 mg (20 μmol) of DTPA-DAB2 is suspended in 0.25 mL N,N Dimethylformamide (DMF) and gently heated to about 50C and sonicated/vortexed to dissolve.
2. 8.33 mL of DDH2O is added to give a cloudy solution that cleared on addition of LnCl3 aqueous solution (0.1 M of LaCl3·6H2O, CeCl3·6H2O, or PrCl3·xH2O; the latter stock solution is dissolved in 0.1 M HCl) with 120 μL of La or Ce solutions or 140 μL of Pr solution, followed by vortexing and bath sonication to give clear light-brown solutions.
3. Aqueous NaOH solution (1 M) is added sequentially in six equal portions (6 × 10 μL) with vortexing after each addition.

(A precipitate is initially formed during the early steps of this neutralization but a mostly clear solution is present by the end).
4. 1.67 mL of 0.3 M sodium cacodylate buffer, pH 7.4 is added, mixed, and centrifuged (3000 × g, 10 min) to remove any precipitate.
5. Solutions are syringe-filtered (0.22 μm, Millipore) immediately prior to addition to cells.
6. Metal ion concentrations can be measured by inductively coupled plasma mass spectroscopy (Agilent 7700).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes a method for screening transgenic lines of <em>Phaeodactylum</em>, specifically for those obtained via conjugation with episomal plasmids.  The assay can also be used for screening genomic integrants obtained through biolistics.  The assay uses patched colonies of diatom grown on agar plates for biomass and does not require the use of kits or any pre-processing (boiling, etc) of the samples prior to analysis.  This allows for relatively high-throughput screening of transgenic lines in a single day.</p>

[STEPS]
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TITLE: A bioinformatics pipeline for processing and analysis of whole transcriptome sequence data
AUTHORS: Ayam Gupta, Shalin Rajagopal, Sonal Gupta, Ashwani Kumar Mishra, Prashanth Suravajhala

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recent advances in next generation sequencing (NGS) technologies have paved the way for augmented genomic research. From the conventional microarray analysis to the current NGS-based whole transcriptome pipelines, there have been significant advances on how differentially expressed genes (DEGs) are identified. With several bioinformatics pipelines in use, inferring the candidate DEGs prove to be a cumbersome approach as one may have to reach consensus in analysing the results from various pipelines. To ensure this, we have benchmarked the well-known Cufflinks-Cuffdiff pipeline on a set of datasets and present it in the form of a protocol for the research community interested in performing downstream analysis of whole transcriptome shotgun sequencing.</div><div class = "text-block"><span style = "font-weight:bold;">Keywords: </span><span>RNA-Seq, Transcriptome analysis, bioinformatics pipeline, differentially expressed genes  </span></div></div>

[STEPS]
TITLE: Cell migration assay or Transwell assay
AUTHORS: Marzia Ognibene

[STEPS]
?. 
?. Cells are washed in cold PBS, detached with 5mM EDTA, centrifuged at 1200 g for 7 min and resuspended in serum-free medium.
?. Transwell chambers (Corning Costar) are prepared filling with 600 μl of complete medium the lower chamber.
?. The desired amount of cells is plated in 100 μl of serum-free medium in the upper insert (8.0 μm pore size) of each Transwell chamber and incubated at 37°C.
?. After 24 hours at 37°C, fill with 600 μl of 5mM EDTA an empty well beside each used Transwell chamber.
?. Eliminate DMEM from the upper insert and move it inside an EDTA well.
?. Collect DMEM from the lower chamber and put some EDTA inside, to detach cells possibly fallen from the upper insert.
?. Detach cells migrated on the underside of the upper insert using a plastic scraper and collecting them in the EDTA well.
?. Mix in one tube cells collected from the lower chamber and scraped from the insert and centrifuge at 1200 g for 7 min, to count the migrated cells.
TITLE: IHC on fixed frozen cryo sections
AUTHORS: Grace Burgin, Inbal Benhar

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

[STEPS]
?. 30-60 min Protein Block 5% Normal Horse (or Donkey) Serum in PBS + 0.3% Triton-X
?. Overnight (~18-22hrs) Primaries at 4C in PBS + 0.3% Triton-X
?. Wash 2x5 min w/ PBS
?. 2hr Secondaries at room temp in PBS + 0.3% Triton-X
?. Wash 2x5 min w/ PBS
?. Coverslip
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the analysis used to plot MEGAN taxonomic assignments. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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TITLE: Coronavirus Lateral Flow Assay (LFA) sample preparation protocol v2
AUTHORS: peijun he

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Coronavirus Lateral Flow Assay (LFA) sample preparation protocol</div></div>

[STEPS]
?. For each clinical sample to be processed, label 1 screwcap tube with the patient’s ID. Make aliquots of Lysis buffer in each tube.Lysis buffer:   in 1x PBS
300 µl
[Triton X - 100]
?. Take out the swabs out of the  freezer immediately before the analysis. Leave to thaw at RT for  and insert into the respective screw-cap tube. Note: handling times within a CL3 lab may be longerthan expected, so do not take the swabs out of the freezer until all tubes are properly labelled andcontaining  Lysis buffer in them.
-80 °C
400 µl
?. ‘Plunge’ each swab up and down in Lysis Buffer for , constantly ‘twisting’ the swab so itssides are brushed against the side of the tube. Note: Ideally, do not handle more than 3-4 swabs at a timeso as to spend enough time twisting each of them to ‘encourage’ maximum dissociation of Nucleocapsidprotein from the swab and lysis of virions.
?. After, discard the swabs appropriately (e.g. in a second container that will then need to beautoclaved before disposal), making sure to recover as much volume out of the swab as possible (will be lost as absorbed by the swab foam) and re-cap the tube.
100 µl
?. Samples can be tested immediately by lateral flow device or stored at for further analysis.
-80 °C
?. Lateral flow assay could be performed directly inside a CL3 lab or within a Class I cabinet after decontaminationof the outside of the tubes before taking them out of the CL3 lab. For the lateral flow assay, follow the lateral flow assay operation protocol.
TITLE: Workflow for human placental bulk ATACseq
AUTHORS: Scott   Lindsay-Hewett, Valentina  Stanley, Mana   Parast, Louise Laurent

[DESCRIPTION]
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate bulk ATACseq data from human placenta.

[STEPS]
SECTION: Tissue preparation
1. As soon as possible after Cesarean section or vaginal delivery, prepare tissue according to the following protocol:


Human Placenta Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC


For this protocol, use tissue that has been snap-frozen.
SECTION: Nuclei isolation
2. Isolate nuclei from 4 samples at a time according to the following protocol:

Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq

Proceed to tagmentation immediately.
SECTION: Tagmentation and library generation
3. Perform tagmentation and library generation according to the following protocol:

Tagmentation and library generation for human placental bulk ATACseq

After passing quality control, proceed to sequencing.
SECTION: Sequencing
4. For HuBMAP bulk ATACseq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration. Alignment and peak-calling were performed using the ATAC-seq pipeline within the bcbio Python package.