eamag/protocols-with-steps huggingface dataset:
TITLE: DAB Staining of Fixed Mouse Brain Tissue Sections
AUTHORS: Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
This protocol describes steps for immunohistochemical 1 3,3’-Diaminobenzidine (DAB) staining of free floating fixed mouse brain tissue sections.

[STEPS]
SECTION: Day 1
1. Blocking and primary antibody incubation.
SECTION: Day 1
1.1. Pull all sections into 1x dPBS in 24-well netwells.
SECTION: Day 1
1.2. Wash 3 times with 1x dPBS, 10 mins each.
SECTION: Day 1
1.3. Wash 3 times with 1x TBS, 10 mins each.
SECTION: Day 1
1.4. Quench endogenous peroxidase activity with quenching buffer, 5 mins at RT. Just covering the sections is sufficient, and drain excess buffer by dabbing on kimwipes before and after.
SECTION: Day 1
1.5. Wash 3 times with 1x TBS, 10 mins each.
SECTION: Day 1
1.6. Block in TBS++++ for 75 mins at RT on rocker, 2 mL/well.
SECTION: Day 1
1.7. Incubate in primary antibody diluted in TBS++++ on rocker at RT O/N, 500 µL/well in a new 24-well plate without netwell insert.
SECTION: Day 2
2.1. Wash 3 times with 1x TBS, 5 mins each in netwell insert.
SECTION: Day 2
2.2. Incubate in secondary antibody at a 1:300 dilution in TBS++++ for 2 hrs, 500 µL/well in a 24-well plate without netwell insert.
SECTION: Day 2
2.3. 15 mins prior to end of incubation, prepare ABC complex:
Both buffer A and B are 1:300 dilution in TBS++++
Incubate in RT beaker of water for 30 mins, in drawer
SECTION: Day 2
2.4. Wash 3 times with 1x TBS, 5 mins each in netwell insert.
SECTION: Day 2
2.5. Incubate 2 hours in ABC complex in TBS++++ at RT on rocker without netwell insert.
SECTION: Day 2
2.6. Wash 3 times with 0.1 M Tris buffer, pH 8.0, 5 mins each in netwell insert.
SECTION: Day 2
2.7. Prepare 1x DAB solution:
Thaw aliquots of 50x DAB, 1 tube is around 200 µL
Add 50x DAB into 0.1M Tris buffer
Vortex for 5 sec to mix well, sit in dark for 10 mins
Right before development, add 30% H2O2 (1:10000 dilution), vortex to mix well
Distribute 2 mL to each well on a 12-well plate
SECTION: Day 2
2.9. Develop DAB reaction, minimizing time difference between wells. Drain excess buffer by dabbing on kimwipes before and after.
SECTION: Day 2
2.10. Wash 2 times with Tris, 5 mins each.
SECTION: Day 2
2.11. Wash 2 times with TBS, 5 mins each.
SECTION: Day 2
2.12. Wash 2 times with PBS, 10 mins each.
SECTION: Day 2
2.13. Sections can be stored in PBS at 4C.
SECTION: Day 2
2.14. Mount onto Superfrost Plus slides labeled with pencil and allow to dry completely.
SECTION: Day 3
3.1. Wash 2 x 2min in ddH2O.
SECTION: Day 3
3.2. Wash 2 x 2min in 70% EtOH.
SECTION: Day 3
3.3. Wash 2 x 2min in 95% EtOH.
SECTION: Day 3
3.4. Wash 2 x 2min in 100% EtOH.
SECTION: Day 3
3.5. Wash 2 x 2min in Xylene.
SECTION: Day 3
3.6. Coverslip using Permount mounting medium.
SECTION: Day 3
3.7. Let dry in fume hood overnight then store in drawer in slide box until imaging.
SECTION: Day 2
2.8. Prepare an additional 12-well plate with Tris buffer to start washes immediately after development.
SECTION: Day 2
2. Secondary antibody incubation and DAB development
SECTION: Day 3
3. Dehydration and clearing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Media for V. natriegens

LB2:
Standard LB media (10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl) with additional 1.5% NaCl


LBv2 Salts:
Standard LB media (10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl) with additional with additional v2 salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM
MgCl2)

BHIv2:
Brain heart infusion Broth (37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500) with additional v2 salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) 
note: BHI can not be autoclaved together with the salts, they have to be autoclave separate and later on added.


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Matthew T Weinstock, Eric D Hesek, Christopher M Wilson, Daniel G Gibson
Vibrio natriegens as a fast-growing host for molecular biology
Nature Methods volume 13, pages 849–851 (2016)

[STEPS]
TITLE: Pull-down Assays
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Pull-down Assays

[STEPS]
1. To pull down proteins from brain lysates and LP2(P4) fractions, 1-2 mg of the
sample was incubated with 25-50 μg of glutathione beads containing GST fusion
proteins for 12-16 hrs. 










2. The mixtures were washed three times with 1X PBS with
0.15% Triton X-100, and then resuspended in 20 μl of 1X NuPAGE LDS sample
buffer for NuPAGE and immunoblotted analysis. 
3. Alternatively, the samples were
resuspended in 50μl of PBS for proteomics studies and stored at -20 °C before
quality control and proteomics analysis.
TITLE: Purification of FIP200 CTR-GFP
AUTHORS: Elias Adriaenssens, Eleonora Turco

[DESCRIPTION]
This protocol details the purification GFP-FIP200 (CTR).

[STEPS]
SECTION: Purification procedure
1. To purify GFP-FIP200(CTR), as described previously (Turco et al. 2019 Mol Cell, PMID: 30853400), fuse the C-terminal domain of FIP200 (1458-1594aa)  to a N-terminal 6xHis-TEV-GFP-tag through cloning into a pET-DUET1 vector (available on Addgene). 

SECTION: Purification procedure
2. After the transformation of the pET-DUET1 vector encoding 6xHis-TEV-GFP-FIP200(CTR) in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C. 

SECTION: Purification procedure
3. Once the cells reached an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min  at  18 °C. 

SECTION: Purification procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer, complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)). 

 Lysis buffer:
 
 
SECTION: Purification procedure
5. Sonicate cell lysates twice for 30 s. 

SECTION: Purification procedure
6. Clear lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
7. Filter the supernatant through an 0.45 µm filter and loaded onto a pre-equilibrated 5 ml His-Trap HP column (Cytiva). 

SECTION: Purification procedure
8. After binding of His tagged proteins to the column, wash the column with three column volumes of wash buffer.

Wash buffer:
 
 
SECTION: Purification procedure
9. Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM). 

SECTION: Purification procedure
10. Pool and incuabte the fractions containing the 6xHis-TEV-GFP-FIP200(CTR) 30 s with TEV protease at 4 °C. 

SECTION: Purification procedure
11. After cleave off the 6xHis tag , recapture 6xHis tag and His-tagged TEV protease with nickel beads for 60 min at 4 °C. 

SECTION: Purification procedure
12. Pellet the beads by centrifugation and the supernatant, concentrate containing the GFP-FIP200(CTR) protein using a 30 kDa cut-off Amicon filter (Merck Millipore) and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
13. Elute the proteins with SEC buffer. 

SEC buffer:
 
 
SECTION: Purification procedure
14. Analyse the fractions by SDS-PAGE and Coomassie staining. 
SECTION: Purification procedure
16. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
17. Store proteins at -80 °C.

SECTION: Purification procedure
15. Pool fractions containing purified GFP-FIP200(CTR).
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Notes:</strong><br /><br />Goal initially is to screen several seawater samples on several roseobacter hosts to determine breadth of rosephage present at each location and then to focus on a single site.<br /><br />Nomenclature for viruses should include a designation for the site of seawater collection followed by a number and the host it was isolated on.<br /><br />Put information into an Excel worksheet. Create a separate Excel file for each source of virus. Use a different tab in each worksheet for different hosts. <br /><br />Turbid plaques indicate possible lysogenic phage that may be of interest to Feng Chen&rsquo;s laboratory in Maryland. Keep the plaque plug at 4&deg;C. Take 50-100 &micro;l from a few and grow up small 1 ml cultures to cryopreserve (glycerol) them. Here trying to get the putative lysogenic cells (prophage containing confers resistance to colonies of cells within the plaque = turbid).&nbsp;

[STEPS]
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TITLE: Spray inoculation of peach trees or leaves with the plant pathogen Xanthomonas arboricola pv. pruni.
AUTHORS: Daniela Negrete-Moreno, Annabel Miller, Elizabeth Cieniewicz

[DESCRIPTION]
Description: 
Potted tree experiments and detached leaf assays are used for many purposes, including: 
    observing the effects of a single pathogen on a host cultivar, 
    observing the effects of multiple pathogen interactions on a host cultivar
    comparing the defense responses and tolerance of different cultivars to pathogen attack.

Abstract:
South Carolina is the leading peach-producing state in the southeastern United States, where multiple pests and pathogens regularly threaten orchards. Here, we describe a methodology for controlled inoculations of potted peach trees or detached peach leaves with the bacterium Xanthomonas arboricola pv. pruni (Xap). This protocol is used primarily to study interactions between Xap and viruses in peach. To further understand the effects of a bacterial-viral co-infection, detached leaf assays serve as a controlled representation of what happens in the field. Hence, we report developing a Xap-specific spray inoculation method that inoculates a medium directly from bacterial colonies. 
 

[STEPS]
1. 
2.
TITLE: Deprescribing criteria for older people
AUTHORS: rdelassalas

[DESCRIPTION]
Introduction/Objective: To propose deprescribing criteria in older people with non-communicable disease (NCD).

Methods: This project was carried out following four stages.
1. A systematic review of the literature was conducted to identify useful tools in the deprescription process.
2. A cross sectional restrospective study of a cohort of more than 30,000 Colombian older people with NCDs was carried out, to whom the Beers criteria were applied.
3. The facial and content validity of an algorithm and a list of criteria for the deprescription of medications were assessed, using the ratio and content validity index and Fleiss's kappa.
4. An approximation of the potential efficiency of deprescription for the health system will be determined through an analysis of cost minimization and budgetary impact of insulin glargine compared to NPH.

Results: A total of 63 tools, 50 explicit criteria and 13 algorithms were identified from the literature review.
The use of the Beers criteria in the patient cohort showed that the most frequent potentially inappropriate prescription is replaced with proton pump inhibitors (23.39%). The algorithm validated by 18 experts from different countries (Colombia, Spain, Argentina) incorporates many questions that attend to the actual clinical practice. The criteria list have 5 recomendations and 33 pharmacotherapeutic groups. The cost minimization analysis of deprescribing of a single drug showed savings and greater efficiency of health system resources.

Conclusion: The proposed algorithm and criteria are tools adjusted to the Colombian context with an international approach data that may be useful to support deprescribing in older people.


[STEPS]
TITLE: Treatment of coagulopathy in COVID-19 patients: protocol for a scoping review
AUTHORS: Hirotaka Mori, Hiroshi Ohkawara, Takayuki Ikezoe, Ryuichi Togawa, Mami Rikimaru, Yoko Shibata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background</div><div class = "text-block">Coronavirus infection disease 2019 (COVID-19) patients have a significant association with coagulopathy and disseminated intravascular coagulation contributing to high mortality. The treatment of COVID-19 and the control of coagulopathy are the primary concern. Although the evidence of anticoagulants in COVID-19 patients with coagulopathy is compiling, there is no scoping review to systematically map the anticoagulants used in this area and to identify the existing gaps between investigated treatment and not. The objective is to conduct a scoping review of available evidence on the anticoagulants in COVID-19 patients with coagulopathy.</div><div class = "text-block">Methods </div><div class = "text-block">For conducting the scoping review protocol, we will use a scoping review methodology in the Joanna Briggs Institute manual. This methodology has a five-stage approach: </div><div class = "text-block">(1) identify the research question; (2) identify relevant studies; (3) select studies; (4) chart the data and (5) collating, summarize and report the results. We also will use the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews to assess the results. </div><div class = "text-block">Ethics and dissemination</div><div class = "text-block">Because this is a review of the literature, ethics approval is not indicated. We will disseminate the findings from this review in publications in peer-reviewed journals. </div></div>

[STEPS]
TITLE: Mitochondrial genome assembly
AUTHORS: Graham Etherington

[DESCRIPTION]
De novo assembly of 49 mustelid whole mitochondrial genomes

[STEPS]
1. Calculate read length of fastq files for each sample and run MitoZ


 

2. Genome alignment. Concatenate the genomes and use ClustalW to align them.

2.1. Rename both the accession name and file name of the genome assemblies, as they'll all have the same name (work71.mitogenome.fa.reorder)

 

2.2. Concatenate all of the assemblies

 
You may need to visualise the genomes to make sure they're all the same complement. Reverse complement any genomes as required.
2.3. Align the assemblies and change format to FASTA
TITLE: HyPR Protocol
AUTHORS: Jamie Marshall, Benjamin R. Doughty, Vidya Subramanian, Philine Guckelberger, Qingbo Wang, Linlin M. Chen, Samuel G. Rodriques, Kaite Zhang, Charles P. Fulco, Joseph Nasser, Elizabeth J. Grinkevich, Teia Noel, Sarah Mangiameli, Anna Greka, Eric S. Lander, Fei Chen, Jesse Engreitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of up to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq, making HyPR-seq a powerful method for targeted RNA profiling in single cells.</div></div>

[STEPS]
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TITLE: Image processing and 3D reconstruction
AUTHORS: Minghao Chen

[DESCRIPTION]
Image processing and 3D reconstruction

[STEPS]
SECTION: Image processing
1. Use cryoSPARC for the following steps except those particularly mentioned. 
SECTION: Image processing
2. Do motion correction by [Patch Motion Correction]
Bin 2x in fourier cropping for super-resolution video stacks
Bin 1x in fourier cropping for regular video stacks
SECTION: Image processing
3. Do contrast transfer function determination by [Patch CTF Estimation]
Remove the outlier micrographs base on the estimated defocus and resolution value.  
SECTION: Image processing
4. Do particle picking by [Topaz]
Manually pick 10 micrographs as learning dataset
Optimize the 'picking threshold' with the 10 mics
Apply the parameter to the entile dataset
SECTION: Image processing
5. Particle extraction
Use the box size 1.5 times larger than the target particles
Bin 4x to facilitate the following classification jobs
SECTION: Image processing
6. 2D classification
Set 50-100 classes dependent on the data size
Remove the obvious junk particles
SECTION: 3D reconstruction
10. Do 3D reconstruction by [Homogeneous Refinement]
Repeat 2-3 times until the resolution converges
Check whether the FSC curve is healthy
SECTION: Image processing
7. Obtain an initial model
[1] Use Ab-initial (Optional) only select the 2D classes that show high-resolution features
[2] Use previously determined structure if it's available
[3] Create a new medel by AlphaFold
SECTION: 3D reconstruction
11. (Optional)
Do CTF refiment followed by homogeneous refinement.
Check whether the resolution get improved
SECTION: Image processing
8. Do 3D classification by [Reterogeneous Refinement]
Low-pass your model to 15-20 Å 
Run the job with 2-3 junk models
Run multiple times (typically 2-4 rounds) until the result converges  
SECTION: Image processing
9. Re-extract the particles with
bin 2x for super-resolution video stacks
bin 1x for regular video stacks
SECTION: 3D reconstruction
12. (Optional)
Do local refinement if the map contains multiple rigid sub-regions
Decide the masks based on [3D Variability] or [3D Flex]
Use [ChimeraX] to create the maps
Use [EMAN2] to compose the final maps at the end
TITLE: DNA extraction and detection of Mansonella perstans from Dry Blood Spots (DBS)
AUTHORS: Millicent Opoku, Dziedzom De Souza

[DESCRIPTION]
Mansonella perstans is a filarial parasite that causes mansonellosis in humans. Unlike other filarial parasitic diseases, mansonellosis is rarely symptomatic and hence poorly studied. This protocol describes the detection of M. perstans in blood. Firstly, DNA was extracted from dried blood blots (DBS). The DNA were screened by a nested PCR and subsequently sanger sequencing was done on excised gels. The sequences were then compared to reference sequences deposited in NCBI. 

[STEPS]
TITLE: Title: Ultrasound-Guided Dry Needling versus Traditional Dry Needling for patients with Knee Osteoarthritis: A Double-blind Randomized Controlled Trial
AUTHORS: Johnson C.Y. Pang, Professor Amy S.N. FU, Dr. Stanley K.H. LAM, Dr. B. PENG, Dr. Allan C.L. FU

[DESCRIPTION]
Abstract
OBJECTIVE: To compare the effect of ultrasound (US)-guided dry needling (DN) with traditional DN in the treatment of pain and dysfunction for patients with knee osteoarthritis (KOA).

DESIGN: A double-blind, randomized controlled trial.

METHODS: Patients (25 male and 65 female), age 50-80 years diagnosed with KOA were recruited and randomly assigned to one of three groups in a 1:1:1 ratio for intervention: real US-guided DN with exercise therapy (G1), placebo US-guided DN with exercise therapy (G2), and exercise therapy solely (G3). G1 and G2 were blinded to the application of real or placebo US guidance by turning the monitor of US imaging out-of-view from participants’ vantage points. The effectiveness of blinding was evaluated by asking the participants whether they had received real-US guided DN. The responses were assessed by Chi-square test. Visual Analogue Scale (VAS), Knee injury, and Osteoarthritis Outcome Score (KOOS) subscales (KOOS-pain, KOOS-symptoms, KOOS-quality-of-life (QoL)) were collected at baseline, 4 weeks, and 8 weeks by a blinded assessor. Data were analyzed by mixed model analysis of variance (ANOVA) with Bonferroni correction.

RESULTS: Eighty-four participants (61.26±5.57 years) completed the study. G1 achieved significant improvement in VAS at 8 weeks compared to G2 and G3 (G1 vs. G2: MD=-15.61, 95% CI [-25.49, -5.51], p=0.001; G1 vs. G3: MD=-19.90, 95% CI [-29.71, -10.08], p< 0.001). G1 achieved significant improvement in KOOS-pain at 8 weeks compared to G2 and G3 (G1 vs. G2: MD=9.76, 95% CI [2.38, 17.14], p=0.006; G1 vs. G3: MD=9.48, 95% CI [2.31, 16.66], p=0.010). KOOS-symptoms and KOOS-QoL were not statistically significant between groups. G2 had no significant difference of the perceptions as G1 with p=0.128. G2 were successfully blinded to placebo US-guided DN.

CONCLUSION: US-guided DN with exercise therapy may be more effective than traditional DN with exercise therapy or exercise therapy alone in reduce pain of KOA.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for quantification of circulating microRNA using TaqMan assays.</p>
<p>MicroRNAs are purified from platelet-poor plasma using Nucleospin columns</p>
<p>During the purification step samples are spiked with cel-miR-39 as a mean of technical normalization</p>
<p>Normalization is performed using the ΔCt-method</p>

[BEFORE_START]
<p>We recommend to use a few samples to test if the volume of spike-in added during microRNA purification is adequate before purifing all your samples.</p>
<p>The Ct-values of the spike-in and the target miRNA should be within the same range, otherwise adjust the volume of spike-in added during miRNA purification.</p>

[GUIDELINES]
<p>Blood samples should be obtained using a minimum of venous stasis and with discard of the first 3 mL of blood.</p>
<p>Platelet-poor plasma should be prepared within 2 hours from blood sampling</p>

[STEPS]
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TITLE: Preparation of 2% Agarose Gel for Electrophoresis
AUTHORS: Stephane  Fadanka, Shalo  Minette, Nadine    Mowoh

[DESCRIPTION]
The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon the charge, size and shape.Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins.Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits.The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. 

[STEPS]
SECTION: Measuring Agarose powder
1. Accurately weigh 0.5 g of  Agarose- electrophoresis grade (Cas 9012-36-6) into a weighing boat using a clean spatula and transfer the powder into a 50ml or 100ml beaker.

SECTION: Measuring Agarose powder
2. Measure 25 mL of 1xTBE  (diluted from a 10x stock as described in the citation below )and dispense into the beaker 
 


SECTION: Measuring Agarose powder
3. Mix by swirling gently and transfer the beaker into the microwave at medium high to heat and boil for 1 min

SECTION: Measuring Agarose powder
4. Turn off the microwave and take out the beaker , allow it to cool for 1 min atRoom temperature, just enough that you can conveniently hold the beaker in your palm.

SECTION: Measuring Agarose powder
5. Pipette 2.5 µLof nucleic acid gel stain (10000x) into the gel while it's still liquid and swirl gently to mix
 


SECTION: Measuring Agarose powder
6. Assemble the electrophoretic gel casting tray on a level surface and put the combs in place
SECTION: Measuring Agarose powder
7. Pour the gel gently into the tray to avoid bubbles
SECTION: Measuring Agarose powder
8. Leave gel to set for 20 min to polymerise

SECTION: Measuring Agarose powder
9. Carefully remove the comb from the solidified gel to have the sample wells
SECTION: Measuring Agarose powder
10. Transfer the gel tray with the solidified gel to a horizontal electrophoretic gel tank and add about 35 mLof TBE buffer (depending on the size of your gel tank) to completely cover the gel. Load the nucleic acid samples into the wells and allow them run.