eamag/protocols-with-steps huggingface dataset:
TITLE: Microalgae fixation for FCM
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for fixing microalgae for later enumeration by flow cytometry.</div></div>

[STEPS]
?. [Preparation]
Aliquot  of 50% gluteraldehyde into each of 3 cryovials.
2 µl
Use a fume hood for this step.
?. [Sample fixation]
Add  of microalgal cell culture sample to each of the 3 cryovials for a final concentration of  gluteraldehyde.
198 µl
?. [Sample fixation]
Mix the sample with the gluteraldehyde by flicking the tube up to 15 times.
?. [Sample fixation]
Incubate tube at  in the dark for .
4 °C
?. [Sample fixation]
Flash freeze the tube in liquid nitrogen.
Use appropriate protective clothing when handling liquid nitrogen.
?. [Sample fixation]
Store at  until ready for FCM.
-80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
-I prepare 8% PFA (w/v) because I like to fix cells without even removing medium (because of the microtubules). If you want to use it 4% you can just dilute 1:1 with fresh PBS1X.<br /><br />- Usually prepare about 250ml batches each time so you'd need ~20g of paraformaldehyde. Both PFAs we have in the lab will work just fine; I prefer the posh one (TAAB pellets) because they are slightly higher grade and they do not form dust (very toxic). But the Sigma powder will work just fine as well. If you use Sigma powder, you should weigh it inside the fume hood too.<br /><br />- I first dilute and rise the pH to solubilize just in water. You may add a shorter volume (~150ml) to rinse later, after adding the pH correcting volumes. Remember that you'll also need a 10% volume (25ml in this example) of PBS10X at the end of the protocol!. Of course use mQ water. The mix will be very milky and acid at the start. Prepare in a suitable glass flask, I usually use a 2X volume Erlenmeyer.<br /><br />- Start mixing and heating moderately inside the fume hood. You may put a parafilm "cap" to minimise fumes and evaporation. Overheating (&gt;55degrees) is to be avoided, use a thermometer if you don't trust your hand. If you play properly with pH, you won't have any trouble and won't need much heating, not even when using the pellets.<br /><br />- Add NaOH (I would recommend high concentrations, 10N) drop wise. Probably you'll need to add about 2mls until the soution clears. It usually takes about 20-30min. so be patient and wait for a while every time you add a small 250microl volume or so. <br /><br />- Once cleared, you can adjust the pH back from the very high (&gt;10) you've got now, to what it should be (7-8), using HCl. Usually you need about 1ml of 1M HCl.<br /><br />- Once adjusted, you can rinse the total volume adding your 10% V of PBS10X and H2O.<br /><br />- Let cool before aliquoting and freezing. I usually freeze it as it is, and then always, before use upon thawing, I filter it with a .45micron. It's very normal after readjusting pH to have some tiny "fibbers" or aggregates; they won't change upon removal the effect of your fixation, but they will be a source of a lot of trouble (very bright autofluorescence, pH goes acid if they stay) if you don't filter.

[STEPS]
TITLE: Anxiety, depression and associated factors among patients with epilepsy attending at outpatients of Ayder Referral, Mekelle and Adigrat Hospitals, Tigray, Ethiopia: A cross - sectional study
AUTHORS: Gessessew Teklebrhan Gebrehiwot, Bizuneh Tesfaye Molla, Asrat Chaka Gizaw, Yodit Habtamu Bezabh

[DESCRIPTION]
Introduction: Depression and anxiety are the most common mental illness, but both are frequently unrecognized and unmanaged among patients with epilepsy. Objective: To assess magnitude of anxiety, depression and associated factors among patients with epilepsy attending at outpatients of Ayder Referral, Mekelle and Adigrat Hospital, Tigray, Ethiopia, 2018. Methods: An institution based cross - sectional study design using was conducted from May 15 to June 24, 2018. Data were collected by using face to face interview and systematic sampling was used. Depression and anxiety were measured by using Hospital Anxiety and Depression Scale (HADS). Binary logistic regression was used to see the association between dependent and independent variables. Results: A total of 416 study participants were interviewed with 99.3% response rate and 59.6% of study participants were male. The mean age of the study participants was 33.19 ±13.66 years. The prevalence of depression and anxiety among people with epilepsy was 37.7 % and 39.9 %, respectively. Depression was associated with poor social support (AOR = 3.33), low monthly income (AOR = 2.17), perceived stigma (AOR =5.01), medication non –adherence (AOR =2.06), side effects of AEDs (AOR = 3.71) and current use of alcohol (AOR =2.35). Anxiety was associated with poor social support (AOR = 4.59), perceived stigma (AOR =2.42), medication non –adherence (AOR =2.77), >= 4 times frequency of seizure attacks per months (AOR = 3.04), and side effects of AEDs (AOR = 4.92). Conclusion: The prevalence of depression and anxiety among people with epilepsy was high. Both depression and anxiety had a statistically significant association with perceived stigma, AEDs side effects, medication non-adherence, and social support. Being lower monthly income and current use of alcohol had a statistically significant association with depression only. A frequency of seizure attacks per months had statistically significant association with anxiety only.
 
 
 
 

[STEPS]
1.
TITLE: Library preparation from a single amplicon pool
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a subprotocol for generating a library from a single amplicon pool</div></div>

[STEPS]
?. Set up the following reaction for each sample:Component                                          VolumeDNA amplicons (5ng/ul)                    Nuclease-free water                           Ultra II End Prep Reaction Buffer     Ultra II End Prep Enzyme Mix           Total
10 µl
2.5 µl
1.75 µl
0.75 µl
15 µl
?. Incubate at room temperature for  Incubate at  for Incubate on ice for
65 °C
?. Quantify the barcoded amplicon pools using the Quantus Fluorometer using the ONE dsDNA assay.
?. Set up the following AMII adapter ligation reaction:Component                                                                    VolumeEnd-repaired amplicon pools                                      Ligation Buffer (LNB)                                                   Adapter Mix (AMX)                                                       Quick T4 DNA Ligase                                                   Total
30 µl
10 µl
5 µl
5 µl
50 µl
There will be some variation in clean-up efficiencies but expect to carry around 80% through a clean-up.
?. Incubate at room temperature for
?. Add (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
50 µl
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add  SFB and resuspend beads completely by pipette mixing.
250 µl
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Remove supernatant and discard.
?. Repeat steps 14-16 to perform a second SFB wash.
?. Pulse centrifuge and remove any residual SFB.
You do not need to allow to air dry with SFB washes.
?. Add EB and resuspend beads by pipette mixing.
15 µl
?. Incubate at room temperature for .
?. Place on magnetic rack.
?. Transfer final library to a new 1.5mL Eppendorf tube.
?. Clean-up end-repair reaction using 1x SPRI beads
TITLE: Discharge preparation and discharge readiness in facilities prior to discharge after birth: a scoping review of the global policies, guidelines and literature
AUTHORS: Helen  Smith, portelaa , Chloe Harvey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Despite existence of global recommendations on postnatal care of mothers and newborns, care provided following childbirth is limited and often fails to address mothers’ concerns about herself and her baby. Women report leaving facilities without sufficient knowledge or skills to take care of themselves and their newborns. Women experience physical and emotional changes during the postnatal period and understanding women’s needs at this time is important for her subsequent health and the health of the newborn. Successful discharge happens when service providers meet the identified postnatal needs of women, caregivers and newborns.</div><div class = "text-block">The World Health Organization is currently developing guidance on postnatal care. One aspect of the guidance relates to readiness for women and their newborns to be discharged from a facility after birth and the steps the health providers should take to prepare a woman, her newborn and parents/caregivers for discharge. However not much is known about the current state of discharge preparation and readiness practices in order to make recommendations for global implementation.As yet there is no comprehensive map of the evidence base; very little is known about working definitions and conceptual boundaries, what criteria for discharge readiness are in use or what strategies or interventions exist for improving discharge preparation and readiness.</div><div class = "text-block">This scoping review will identify and map the range of policies, guidelines and literature on discharge readiness, clarify definitions of the concepts of discharge preparedness and discharge readiness, list criteria and items currently used to assess discharge readiness, and report on any tools, processes, strategies or other interventions that have been developed and/or implemented to improve discharge preparation and readiness.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Enzyme solution for the isolation of protoplasts suitable for <em>Arabidopsis thaliana</em> and <em>Nicotiana benthamiana</em>. Adapted from Yoo et al. 2007 http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.html</p>
<p>And excellent video from the Sheen Lab is available: https://www.youtube.com/watch?v=5-xm1EoLrW4</p>
<p> </p>

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

[DESCRIPTION]
<p>ADAMTS5, the main aggrecanase, is a protein that seems to play a functional role in the development of brown and white adipose tissue (WAT) and browning of WAT. These observations were made using the <span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">ADAMTS5-P mice originally generated by Pfizer, in collaboration with Lexicon Genetics (The Woodlands, TX, USA). ADAMTS5-J mice were generated by Deltagen Inc. and are now commercially available through The Jackson Laboratory (Bar Harbor, USA). The targeting construct, containing an IRES-lacZ-neomycin cassette, substituted 134 nucleotides of exon 2. </span></p>

[GUIDELINES]
<p>-Tails digested in lysis buffer containing proteinase K can be stored at -20°C before continuing the protocol.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Universal sandwich enzyme linked immunosorbent assay for investigating   protein-AG (SpAG)  interactions with immunoglobulins using a SpL-HRP conjugate.
AUTHORS: Angel  Justiz-Vaillant, Norma McFarlane-Anderson

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

[STEPS]
?. This ELISA is used to study the interaction of a protein-AG (SpAG) with different immunoglobulin preparations from mammalian species.
?. The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of a mixture of protein-A and protein-G 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  animal serum (1 mg/ml) is added and incubated for 1h at room temperature and the microplate is rewashed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled SpL conjugate diluted 1:3000 in PBS-non-fat milk is added to each well and  incubated for 1h at RT. The plate is washed 4X with PBS-Tween.
?. 50 µl of 4 mg/ml o-phenylenediamine solution (OPD) is added and the plate is incubated 15 minutes at RT in the dark.
?. 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 492 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the affinity of SpAG to mammalian immunoglobulins.
TITLE: GCase activity in mouse brain samples
AUTHORS: Pietro La Vitola

[DESCRIPTION]
This protocol is designed for assessing the beta-glucocerebrosidase activity in fresh collected mouse brain samples

[STEPS]
SECTION: Samples homogenization
1. Homogenize fresh collected mouse brain samples in  lysis buffer (50 mM Tris-HCl, pH 7.4 and 750 mM NaCl, 5 mM EDTA and 10%Triton X-100). 
SECTION: Samples homogenization
2. Centrifuge samples at 5000 g (10 min at 4 °C ) 
SECTION: Samples homogenization
3. Collect the supernatant
SECTION: Samples homogenization
4. Measure protein concentration with the Pierce BCA Protein Assay Kit (Fisher Scientific, 23225). 
SECTION: Assesment of GCase activity
5. Make a serial dilution of 4-Methylumbelliferone (M1381, Sigma) dissolved in 1% Methanol ddH20
SECTION: Assesment of GCase activity
6. Put 10µL of each standard dilution and of each sample in a pre-prepared black 1.5 ml tube (in duplicate or triplicate). 
SECTION: Assesment of GCase activity
7. Add 25µL of Reaction Buffer and 65µL of Substrate buffer in each tube
SECTION: Assesment of GCase activity
8.  Incubate60 min at 37 °C 

SECTION: Assesment of GCase activity
9. Remove samples from the incubator and add 90µL of Stopping buffer to each tube 
SECTION: Assesment of GCase activity
10. Load 150µL of all samples, standards and blank (35µL of Reaction Buffer + 65µL of Substrate buffer + 90µL of Stopping buffer) into a 96 well black plate 
SECTION: Assesment of GCase activity
11. Measure fluorescence using plate reader (Excitation at 365 nm and emission at 450 nm)
SECTION: Assesment of GCase activity
12. Calculate the amount of 4-Methylumbelliferone  generated in each sample based on the standard curve 
SECTION: Assesment of GCase activity
13. Normalize the data obtained based on the amount of protein (mg) that were in each reaction tube
TITLE: Non-radioactive phosphorylation with T4 PNK (M0201)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Protocol for non-radioactive phosphorylation with T4 PNK.

[STEPS]
1. Set up the following reaction in a microcentrifuge tube on ice:
 

 

2. Incubate at 37 °C for 30 min.
3. Heat inactivate by incubating at 65 °C for 20 min.
TITLE: Patient Satisfaction Survey of the Knee Surgery Project
AUTHORS: Maressa  Lima, Fabricio Loures, Guilherme  Runco, José  Maurício Moraes do Carmo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To evaluate the satisfaction rate of patients who underwent surgical treatment of total knee arthroplasty at Hopstial Universitário Pedro Ernesto.</div></div>

[STEPS]
?. To raise the files of all patients who underwent total knee arthroplasty at the Pedro Ernesto University Hospital in 2020.
?. Make telephone contact and explain the content and importance of the research to improve the quality of assistance
?. Conduct the interview with nine questions for patients who agree to participate.
?. Compile the data.
TITLE: Phylogenetic analysis
AUTHORS: Joachim Nwezeobi, Onyeyirichi Onyegbule, Chukwuemeka Nkere, Joseph Onyeka, Sharon van Brunschot, Susan Seal, John Colvin

[STEPS]
?. The partial mtCO1 sequences obtained from sequencing two randomly selected whiteflies from each of the 119 locations were first screened using quality checks in Geneious (v10.2.6) and those failing to meet the following criteria were discarded
?. First, the sequence chromatograms were manually checked and translated using the invertebrate mitochondrial genetic code. Poor quality sequences and sequences containing stop codons were excluded and the sequencing process repeated for these samples.
?. Two hundred and thirty-eight contig sequences were assembled from sequence data that passed the quality control and were trimmed to 657 bp and aligned with 691 B. tabaci mtCO1 reference sequences downloaded directly from GenBank.
?. The resulting alignment was used to plot an initial phylogenetic tree that was used to identify the whitefly genetic groups, using the FastTree program in Geneious (v10.2.6).
?. Twenty-nine unique haplotypes were identified in the 238 new sequences, which were extracted with Geneious 10.2.6 and used to generate the main phylogenetic tree. These unique sequences were combined with 18 selected reference sequences and aligned. A mtCO1 sequence of the spiralling whitefly Aleurodicus dispersusRussell (GenBank code: AJ748380) was used as an outgroup for the analysis.
?. The best model of nucleotide sequence evolution was evaluated using jModelTest (v2.1.10)
?. MrBayes (v3.2.6) was run using the nexus-formatted aligned sequences and the Generalised time-reversible (GTR) model [48,49] and Gamma distribution with invariant sites (G+I) as well as with four chains for 50 million generations and the trees were sampled every 1,000 generations.
?. The consensus tree (.nex.con.tre) file was used to view the phylogenetic tree and the posterior probabilities of the branches using FigTree (v1.4.3) [50]. The tree was further annotated using Adobe Illustrator (v22.1). The 29 unique haplotype sequences were submitted to the GenBank sequence database and were each assigned unique accession numbers.
TITLE: Measuring mitophagy via FACS with mtKeima reporter
AUTHORS: Louise Uoselis

[DESCRIPTION]
Preparation of samples for measuring mitophagy levels using mtKeima reporter by fluorescence activated cell sorting (FACS).

[STEPS]
SECTION: Day 1
1. Seed cells in a 24 well plate, aiming for a confluency of ~80-90% at the time of treatment. Seed additional wells of cells not expressing any fluorescent proteins, cells expressing only mtKeima, and cells expressing only YFP-Parkin (to be used as gating controls).
SECTION: Day 2
2. Feed all cells with standard growth media for 60 min prior to treatment.

SECTION: Day 2
3. Replace media in each well with media containing the drug you are treating with.
NOTE: Do not change the media or treat the additional wells of cells to be used for gating control.
SECTION: Day 2
4. 60 min prior to harvesting, feed the untreated wells with 0.5 mLof fresh growth media. 

SECTION: Day 2
5. At the conclusion of the treatment time point, harvest the cells using the following procedure:
SECTION: Day 2
5.1. Aspirate media from all wells.
SECTION: Day 2
5.2. Wash all wells once with 500 µLof Room temperature PBS.

SECTION: Day 2
5.3. Add 150 µLof trypsin to each well, and incubate cells at 37 °C for 90 s

SECTION: Day 2
5.4. Place plates onto ice, and harvest each sample into a separate microfuge tube on ice by resuspending each sample with 500 µL of ice cold standard growth media.

SECTION: Day 2
6. Centrifuge all samples at 1000 rcf for 90 s at 4 °C

SECTION: Day 2
7. Carefully aspirate the supernatant from all samples.
SECTION: Day 2
8. Resuspend each sample in 50 µL of FACS sorting media (10% v/v FBS, 1 mM EDTA in PBS) and place into FACS analysis tubes. Keep samples on ice until immediately prior to analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol has been very widely used in our laboratory. The local lab labeling is called « amplification of the <em>R0</em> region », However it is clearer to say that the amplicon corresponds to position 912 to 1264 of reference sequence from Kuo et al 1988, excluding the primer sequences. Sens primer is a 22 mer well conserved as locate complementary to the antigenomic ribozyme sequence cleavage site. The sens primer sequence 890-&gt;911 is 5’ CAT gCC gAC CCg AAG AGG AAA g 3’. Antisens primer corresponding to the antigenomic sequence 1288-&gt;1265: 5’ gAA ggA Agg CCC TCg AgA ACA AgA 3’ which is in the HD gene, corresponding to the sequence of the peptide ‘RKALENK’ located at the end of the first arginin rich motif.</p>
<p>As this amplicon also contains the sequence coding the carboxyterminal extention of L-HDAg, it has been widely used for genotyping although caution should be made: HDV genotyping best relies on the full length S-HDAg gene or the full-length genome sequence.</p>

[STEPS]
?.
TITLE: Immunofluorescence free-floating rat brain cryosections
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for immunofluorescence on rat brain cryosections 

[STEPS]
SECTION: 1. Sections selection
1. Collect the cryosections needed for a caudo-rostral representation of each brain region (every fifth or sixth section depending on the section's thickness, brain region, and animal species) into a 24-well-plate (3-4 sections per well)
SECTION: 1. Sections selection
1.1. Wash in PB1X 3 times x 5 min  (500 ul) at RT
SECTION: 2. Blocking
2. Incubate with blocking solution (500 µ l/well) : PB1X + 10%NDS (serum from the same species as the host of the secondary antibody) + 0.3% TritonX-100, at RT
SECTION: 3. Primary antibody incubation
3. Remove the blocking solution and incubate sections with PB1X+ 5%NdS+0.3% TritonX-100+ primary Ab (500 µ l/well) for 24/72 h, depending on the Ab, at +4°C 
SECTION: 3. Primary antibody incubation
3.1. Wash  in PB1X 3 times x 5 min  (500 ul) at RT 
SECTION: 4. Secondary antibody incubation
4. Incubate sections with adequate secondary fluorescent Ab diluted in PB1X+ 0.3% TritonX-100, at RT 
SECTION: 4. Secondary antibody incubation
4.1. Wash  in PB1X 2 times x 5 min  (500 ul) at RT 
SECTION: 5. Section Mounting
6. Mount sections on slides and let it dry 
SECTION: 4. Secondary antibody incubation
5. Incubate with DAPI  diluted in PB1X, to stain nuclei
SECTION: 4. Secondary antibody incubation
5.1. Wash  in PB1X 2 times x 5 min  (500 ul) at RT 
SECTION: 6. Mount Coverslips
7. Coverslip slides with aqueous mounting medium, remove bubbles if any, and let dry
TITLE: Protocol for whitefly genetic analysis
AUTHORS: Joachim Nwezeobi, Onyeyirichi Onyegbule, Chukwuemeka Nkere, Joseph Onyeka, Sharon van Brunschot, Susan Seal, John Colvin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Bemisia tabaci</span><span style = "font-style:italic;font-style:italic;">(</span><span style = "font-style:italic;">sensu latu</span><span>) is a group of >40 highly cryptic whitefly species that are of global agricultural importance, both as crop pests and plant-virus vectors. This protocol outlines the process for extracting genomic DNA from </span><span style = "font-style:italic;">B. tabaci</span><span> whiteflies and the steps taken in analysing the DNA sequences for identifying the identities of the whiteflies collected in eastern Nigeria </span></div></div>

[STEPS]
TITLE: Measurement of insulin concentration
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

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

[STEPS]
?. MIN6 cells were cultured for 30 min in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES, and 0.1% bovine serum albumin) containing 2 mM glucose.
?. The buffer was then changed to another KRBH buffer solution containing the relevant experimental concentration of glucose, and the cells were cultured for 1 h.
?. The KRBH buffer was collected and its insulin concentration was measured.
?. The insulin concentration of the buffer was normalized to the total cell protein level.The results were normalized to the concentration of control samples of each independent experiment and the normalized values were shown as insulin secretion ratio.