eamag/protocols-with-steps huggingface dataset:
TITLE: Midbrain-like Organoids generation from hiPSCs
AUTHORS: Hariam Raji, michela.deleidi

[DESCRIPTION]
In this protocol we describe the differentiation of human induced pluripotent stem cells (hiPSCs) into human midbrain-like organoids (hMLOs). This protocol has been developed based from several published protocols.

[STEPS]
SECTION: Day 0
1. Dissociate iPSC colonies to single cells with Accutase for 7 min at 37 °C.

SECTION: Day 0
2. Re-suspend cells in day0 medium and plate 8.000 cells/well in 96-Wells Plate U-round-Bottom Low Attachment.
SECTION: Day 4
3. Carefully exchange the medium, without touching the EBs.
SECTION: Day 7: Matrigel embedding
4. Dilute Matrigel in a 3:2 ratio with day 4 medium (used as an embedding mixture).
SECTION: Day 7: Matrigel embedding
5. Wash EBs in day 4 medium. Transfer and mix 5-8 EBs into the embedding mixture and plate onto a 6-well ultra-low-attachment plate.
SECTION: Day 7: Matrigel embedding
6. Incubate for 30 min at 37 °C and add day 7 medium.

SECTION: Day 7: Matrigel embedding
7. Incubate at least 1440 min between d7 and d8.

SECTION: Day 8
8. Add fresh differentiation medium without disrupting the embedded EBs.
SECTION: Day 8
9. After Day 8 change medium twice a week. 
SECTION: Day 10-13
10. At day 10 reduce CHIR to 3.0 micromolar (µM) (1:1000).

SECTION: Day 13-15
11. Reduce CHIR to 0.7 micromolar (µM). Remove CHIR from medium at day 15 and onwards.

SECTION: Day 20-25
12. Manually dissociate organoids from Matrigel, using two surgical needles.
SECTION: Day 20-25
13. Place on orbital shaker after dissociation ( ).
TITLE: BAF_Protocol_015_ 3-NPH derivatization of short chain fatty acid
AUTHORS: Nicholas Sherman

[DESCRIPTION]
Short Chain Fatty Acid derivitization before MS analysis.

[STEPS]
SECTION: Samples
1. Refer to BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction for soluble metabolite extraction of a sample of interest and dry the extracted metabolites.

SECTION: Standards and standard curve
7. Prepare a stock solution of each standard at 10 mg/mL in 75% MeOH. Use the stock solution to generate a 1000 ug/mL
SECTION: Standards and standard curve
8.  Prepare standard curve:
 
 
SECTION: Derivatization reaction
9. Solubilize metabolite extracts in 100 µL  of 75% methanol 
Add 50 µL  of each into a clean new Eppendorf tube      

SECTION: Derivatization reagents: prepare all fresh
2. Reagent powder storage: EDC is stored at -20C, all others are at room temperature. 
Prepare working solutions on small glass botles.
SECTION: Derivatization reagents: prepare all fresh
6. 7,5% pyridine in 75% methanol.
--> add 750 uL in 9.25 mL of 75% methanol.
SECTION: Derivatization reagents: prepare all fresh
5.  150 mM EDC (stored at -20C) in 100% methanol:
--> dissolve 28.75 mg in 1 mL methanol. 
SECTION: Derivatization reagents: prepare all fresh
4. 2 mg/mL BHT in 100% methanol:
--> dissolve 2 mg in 1 mL methanol.
SECTION: Derivatization reagents: prepare all fresh
3. 250 nM 3-NPH solution as derivatization agent in 50% methanol:
--> dissolve 47.4 mg of 3-NPH in 1 mL of 50% methanol
SECTION: Derivatization reaction
12. To quench the reaction, add 50 µL  of BHT

SECTION: Derivatization reaction
11. To each sample or standard add:
50 µL  of 150 mM EDC,
50 µL of 7,5% pyridine,
Incubate in thermomixer at 30 ºC
SECTION: Derivatization reaction
10. Add  50 µL  of each standard curve level into a clean new Eppendorf tube

SECTION: Derivatization reaction
13.  Dilute with 250 µL  of water making a total of volume of 500 µL  and to achieve 40% methanol at final sample.

SECTION: Derivatization reaction
14. Samples are ready to be analyzed by LC-MS/MS. Use 40% methanol as blank runs.
SECTION: LC-MS/MS parameters - Vanquish UPLC
15. Column: Acquity UPLC BEH C-18 1.7 um 2.1 x 150 mm (Waters)
Solvent A: 0.1% FA in water
Solvent B: 0.1% FA in 90% methanol
Flow = 0.250 ml/min. 
Gradient: 
0-1 min 50%B; 1-5 min 70% B; 5-8 min 98% B; 8-13 98% B;  13-13.1 min 15%B.
SECTION: Mass spectrometer parameters - Orbitrap ID-X
16. Full scan parameter 
MS1 Res = 120,000
Range = 67-1000
RF Lens = 60
AGC = 25%
Max IT (ms)= 50
Polarity = Negative

PRM parameters:
CE=30
Res: 50,000
Isolation window: 1.5
AGC: 50
MaxIT (ms): 86
Polarity = Negative

Targeted masses:

 

Analyze data using Skyline.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is following RNA-SIP experiments and RNA extraction to fractionate the RNA according to density. The protocol is based on Leuders et al. 2010.</p>

[STEPS]
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TITLE: ADN PURIFICATION FROM AGAROSE GELS WITH GENELUTE GEL EXTRACTION KIT 
AUTHORS: Ana Belem García González, Georgina Elisa Diego Macías, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
Protocol for DNA purification from  DNA purification from agarose gels with GeneElute Gel Extraction Kit

[STEPS]
1. Previous electrophoresis : Realization of an electrophoresis in a agarose gel of the product of ADN, charging the maximum amount of DNA per well (if necessary, use more than one well)
2. Extraction of the band of interest: Extract the band of interest of the gel, removing the excess of agarose
3. Weighting the band: Weight the piece of gel removed in a tarated tube.
4. Gel Solubilization: Add 3 volumes of Gel Solubilization Solution to the piece of gel, meaning, for each 100 mg of agarose gel, add 300 µL of Gel Solubilization Solution. Incubate at 50-60°C for 10 min minutes or until the piece of gel has completely dissolved. Give Vortex every 2 or 3 minutes in this incubation time.

5. Preparation of the column (This can be done alongside step 4). Add GenElute Binding Column G in a recolectation tube of 2 mL. Add 500 µL of the Column Preparation Solution to each Binding Column. Centrifugate for 1 min minute at 12,000 to 16,000 x g. Discard liquid

6. Check the color of the sample: Once the gel had dissolved completely, make sure that the color is yellow before continuing. If the color is red, add 10 µL of the Sodium Acetate 3M, pH 5.2 buffer and mix. Add until the color is yellow.

7. Add isopropanol: Add a volume of the gel of isopropanol at 100% and mix until homogenized.
8. Bind ADN: Charge the sample of step 7 to the binding column prepared in step 5. Is normal a change of color from yellow to red once the sample is charged. If the volume of the sample is bigger than 700 µL, charge the sample in proportions of 700 µL. Centrifugate for one minute at 12,000-16,000 x g  every time after the sample is charged. Discard the liquid obtained from the centrifugation.

9. Wash the column: Add 700 µL of Wash Solution to the binding column. Centrifugate for one minute at 12,000-16,000 x g. Remove the binding column of the tube and discard liquid. Colocate again the binding column in the recollection tube and centrifuge one more time for one minute to eliminate the excess of ethanol

10. Elute the DNA: Transfer the binding column to a clean, esteril tube. Add 50 µL  of the Elution Solution to the center of the membrane and incubate for1 min minute. Centrifuge for 1 min minute at 12,000-16,000 x g. In case of purifying plasmid DNA or linear fragmented of DNA with a weight exceeding 3Kb, preheat the Elute Solution at  65 °C
TITLE: RNA-Isolation from milk samples for virus analysis
AUTHORS: Sudhir Bhatia, Gudrun Baersch

[DESCRIPTION]
This protocol is about a method to isolate RNA from the milk samples to conduct the viral analysis, for example avian Influenza Virus H5N1, Measles, Zika Virus, Dengue Fever Virus etc. in human and other mammalian species. The isolated RNA can be used for conducting the molecular biological analysis like conventional as well as real time PCR, nucleic acid based nanoparticle assays etc. 

[BEFORE_START]
Read the manual and note the important points e.g. temperature and centrifugation speed.


[GUIDELINES]
Read the manual before use. 
Check the contents of the kit. 

Precautions:
- The kit is only for in vitro diagnostic use.
- The kit may only be used by trained persons. 
- The user must read the manual carefully. 
- The kit should not be used after expiry date.
- The user should work very cleanly when isolating.
- Decontaminate the instruments regularly (once a week).
- The user should wear protective gloves and laboratory clothing. 

[STEPS]
1. Adjust the temperature of heat block.
2. Add 300µl of Tube A (Lysis buffer I) and 15µl of Tube K (Proteinase K) to the 100µl milk sample in the tube.
3. Incubate at 56°C for 20-30 minutes. Add to this 400µl of Tube G (Lysis buffer II).
Incubate at 70°C for 5 minutes.
4. Add to this sample 400µl of molecular ethanol and shake with the vortexer.
5. Take a mini column in one collection tube and add 600µl of above made solution to this
mini column.
6. Centrifuge this for one minute at 11000RPM. Discard the filtrated fluid.
7. Add the rest of your remaining fluid in this mini column and repeat the step 5 for centrifuge. Discard the filtrated fluid.
8. Now add 500µl of Tube B (Washing buffer I) to mini column. Repeat the step 5 for centrifugation and discard the filtrated fluid.
9. Add 500µl of Tube C (Washing buffer II) to mini column. Repeat the step 5 for centrifugation and discard the filtrated fluid.
10. Add 200µl of Tube C to mini column. Repeat centrifugation for 3 min at 13000RPM and discard the filtrated fluid.
11. Centrifuge the mini column for 1 min at 13000RPM to dry the matrix. Discard the used collection tube.
12. Now put the mini column (filter part) in a new 1.5 ml collection tube.
13. Add 100µl of Tube E (pre-warmed to 70°C) to the mini column.
14. Now keep this at room temperature for two minutes.
15. Centrifuge this at 13000RPM for one minute.
16. Now the user has fluid in the collection tube. This is isolated RNA. This can be used to perform different assays. The RNA should be stored at -20°C for long-term use.
17.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The mtDNA fragment (15869-740) was amplified using primers for polymerase chain reaction (PCR) : L15869F and H719R (L15869 F 5' AAAATACTCAAATGGGCCTGTC 3', H719R 5' CGTGGTGATTTAGAGGGTGAAC 3'). The 20 µl PCR reactions contained 2.0 µl 5×buffer, 1.6 µl 2.5 mM dNTP mix, 0.8 µl each of reverse (R) and forward (F) PCR primers (8 pM each), 0.2 µl of KOD Enzyme (1.0 U/µl), and 20 ng of template DNA. PCR was performed under the following cycle conditions: initial denaturation of 94 °C for 5 minutes; followed by 35 cycles of 94 °C denaturation for 30 seconds, 55 °C annealing for 30 seconds, and 72 °C elongation for 40 seconds; followed by a final extension at 72 °C for 10 minutes. The production fragment was sequenced with the following primers: L15869F and 80R for HVSI, 16539F and H719R for HVSII. The purified PCR products were sequenced by ABI 377 DNA automatic sequencer.</p>

[STEPS]
TITLE: Effect of lumbar lordosis angle in adolescent baseball players  on the development of lumbar spondylolysis
AUTHORS: Yuki  Onoda, Takashi  Kitagawa

[DESCRIPTION]
Abstract
Background: Lumbar spondylolysis is a stress fracture of the lumbar vertebral arch that occurs frequently in adolescents. Lumbar spondylolysis has a high prevalence in athletes, especially baseball players. When lumbar spondylolysis occurs, restriction of sports activities is inevitable until the bony union is achieved. Therefore, prevention of the onset of lumbar spondylolysis is necessary, and it is necessary to elucidate the risk factors that influence the onset of the disease. An increase in lumbar lordosis angle may influence the development of lumbar spondylolysis because the lumbar lordosis angle increases the compressive stress in the vertebral arch. However, there are no reports on the effect of lumbar lordosis angle and the development of lumbar spondylolysis in adolescent baseball players. Therefore, the purpose of this study was to investigate the effect of lumbar lordosis angle on the development of lumbar spondylolysis in adolescent baseball players.
METHODS: Eligible patients were those who visited the orthopedic clinic from January 1, 2018, to October 31, 2021. The selection criteria were male baseball players aged 11-18 years who visited the clinic, and the exclusion criteria were those whose superior endplate of L1 and superior endplate of S1 could not be identified in the MRI images. The existence of development of lumbar spondylolysis, lumbar lordosis angle, age, and pitching experience of the above patients will be assessed based on electronic medical records and imaging findings. Statistical analysis was performed using logistic regression analysis, with the objective variable being the existence of lumbar spondylolysis and the explanatory variables being the lumbar lordosis angle, age, and previous pitching experience.
Discussion: This study examines the effect of the lumbar lordosis angle on the development of lumbar spondylolysis in adolescent baseball players. An increase in lumbar lordosis angle may influence the development of lumbar spondylolysis and may be a risk factor for the development of lumbar spondylolysis.

[STEPS]
TITLE: UMass - Coronary Artery Ligation
AUTHORS: Richard McIndoe, Mark Kelly, Timothy P. Fitzgibbons

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a mouse model of acute myocardial infarction. This protocol is suitable for studying mouse models thought to be relevant to coronary ischemia or the development of congestive heart failure.</div></div>

[STEPS]
?. Anesthetize the mouse and ensure depth of anesthesia with a toe pinch.
?. Shave the left thoracic area of the mouse.
?. The mouse is intubated using a 22-G angiocatheter sheath and placed on a rodent ventilator with the left chest up, secure feet to surgical area with tape.
?. Prep the surgical field with 70% isopropanol as well as Betadine solution and drape.
?. A left side thoracotomy is performed between the 3rd and 4th intercostal space and opened approximately 6-7 mm with a small retractor. Use caution to avoid injury to the lung.
?. After making an opening the pericardium, the left anterior descending coronary artery is identified using a microscope. A 7-0 Proline suture is placed just under the descending coronary artery without entering the left ventricle. This can be tied permanently to ligate the LAD for an acute ischemia model or alternatively, the 7-0 proline suture is secured over a 3-4 mm piece of PE-90 tubing to ligate the LAD for 30 minutes, and then the suture is removed to facilitate reperfusion of the heart for an ischemia reperfusion model. The suture is placed 2 mm below the left atrium for a “large” infarction; 3 mm below the left atrium for a “medium” infarction; or 4 mm below the left atrium for a small infarction.
?. Two separate 6-0 Proline sutures are place to close the rib cage
?. A 20g iv catheter sheath is left between the sutures to evacuate air after closure is complete.
?. The dermis is sutured closed with 6-0 Proline, Tissue clips or glued with surgical glue
?. Air is removed from thoracic cavity via the sheath and a 1-mL syringe. Gently pinching the skin around the sheath slowly draw back on the syringe while withdrawing the sheath.
?. Animal is weaned off anesthesia and extubated when spontaneous breathing resumes.
?. Injection 0.5 ml of 0.9% NaCl subcutaneously to each mouse to prevent dehydration.
?. Post-procedure Analgesics:Buprenorphine (0.05mg/kg) every 12 hours, for 72 hours post-op.Meloxicam (5mg/kg) every 24 hours, for 72 hours post-opPost-procedure Monitoring:Mice are monitored 2x daily for the first 72 hours after the surgery. Thereafter, mice are monitored at least 3x per week.
TITLE: The efficacy of non-pharmaceutical treatment of menopausal musculoskeletal symptoms: A state-of-the-art scoping review protocol
AUTHORS: Delyth Wyndham, June Keeling

[DESCRIPTION]
Objective: 

The objective of this state-of-the-art scoping review is to examine the scope of the contemporary research literature evaluating the efficacy of non-pharmaceutical treatment of musculoskeletal symptoms during menopause.

Introduction: 

Although recent studies and reviews often reveal the complexities of menopause symptoms, no current reviews were identified that summarise contemporary existing knowledge on the efficacy of the variety of non-pharmaceutical treatments from a symptoms-based perspective.  Menopause has an adverse impact on musculoskeletal health due to reducing oestrogen, which may result in painful symptoms.  The prevalence rates of musculoskeletal symptoms during menopause highlights the need for treatment efficacy.  The efficacy of non-pharmaceutical treatments for musculoskeletal symptoms has not yet been fully explored in a state-of-the-art scoping review.

Inclusion criteria: 

This review will consider cross-sectional, longitudinal, prospective or retrospective studies; reviews; or evidence-based syntheses/guidelines considering the efficacy of a non-pharmaceutical treatment of musculoskeletal symptoms during menopause.  The inclusion criteria for article selection will be primary research presented in full text English language articles published in peer reviewed journals.  During screening it will become apparent the right moment to mark the beginning of state-of-the-art thinking about the topic.

Methods: 

The state-of-the-art scoping review aims to methodically explore the quantity and type of research evaluating the efficacy of non-pharmaceutical treatment of musculoskeletal symptoms during menopause.  The key information sources to be searched are the Cochrane Central Register of Controlled Trials (CENTRAL); EBSCO; Web of Science; and EMBASE (Ovid).  Two reviewers will screen the titles and abstracts for inclusion using the Rayyan research collaboration platform.  The full text of publications meeting the inclusion criteria will be examined for eligibility.  Meta-data will be extracted using a standardised template.


[STEPS]
TITLE: Change in treatment burden among people with multimorbidity: protocol of a follow up survey and development of efficient measurement tools for primary care.
AUTHORS: H.O.Hounkpatin , *, Paul Roderick, James E Morris, James E Morris, Scott Harris, Forbes Watson, Hajira Dambha-Miller, Helen Roberts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Treatment burden is the effort required of patients to look after their health and the impact this has on their functioning and wellbeing. It is likely treatment burden changes over time as circumstances change for patients and health services. However, there are a lack of population-level studies of treatment burden change and factors associated with this change over time. Furthermore, there are currently no practical screening tools for treatment burden in time-pressured clinical settings or at population level.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Methods and analysis</span></div><br/><div class = "text-block">This is a three-year follow-up of a cross-sectional survey of 700+ people with multimorbidity (defined as three or more long-term conditions; LTCs) registered at GP practices in in Dorset, England. The survey will repeat collection of information on treatment burden (using the 10-item Multimorbidity Treatment Burden Questionnaire (MTBQ) and a novel single-item screening tool), sociodemographics, medications, LTCs, health literacy and financial resource, as at baseline. Descriptive statistics will be used to compare change in treatment burden since the baseline survey in 2019 and associations of treatment burden change will be assessed using regression methods. Diagnostic test accuracy metrics will be used to evaluate the single-item treatment burden screening tool using the MTBQ as the gold-standard. Routine primary care data (including demographics, medications, LTCs, and healthcare usage data) will be extracted from medical records for consenting participants. A forward-stepwise, likelihood-ratio logistic regression model building approach will be employed in order to assess the utility of routine data metrics in quantifying treatment burden in comparison to self-reported treatment burden using the MTBQ.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Impact</span></div><br/><div class = "text-block">To the authors’ knowledge, this will be the first study investigating longitudinal aspects of treatment burden. Findings will improve understanding of the extent to which treatment burden changes over time for people with multimorbidity and factors contributing to this change, as well as allowing better identification of people at risk of high treatment burden. </div><br/></div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Mannitol oxidase is an enzyme present in some gastropods, converting the polyalcohol mannitol into the sugar mannose. Molecular oxygen is the hydrogen acceptor and hydrogen peroxide is produced. Therefore, this enzymatic activity can be measured by monitoring H<sub>2</sub>O<sub>2</sub> production using a horseradish peroxidase coupled assay adapted from previously published methods (Malik et al., 1987; Cablé et al., 1993; Rocha et al., 2003). This assay was used to evaluate mannitol oxidase activity in the digestive gland of gastropods, but in some slugs and snails this enzyme was also detected in digestive tract tissues (Vorhaben et al., 1984; Malik et al., 1987). This enzyme can also oxidase other polyalcohols (Large &amp; Connock, 1994)</p>

[BEFORE_START]
<p>Prepare the following solutions:</p>
<p> </p>
<p><strong>Homogenization medium for marine gastropods</strong></p>
<p>(adapted from Stewart et al. 1994 and Moyers et al. 1991)</p>
<p>Sucrose - 500 mM</p>
<p>Potassium chloride - 150 mM</p>
<p>Ethylenediamine tetraacetic acid (EDTA) sodium salt - 1 mM</p>
<p>Tris-HCl 50 mM pH 7.4</p>
<p> </p>
<p>This medium, without phenylmethylsulfonyl fluoride (PMSF), can be stored at -20º C in aliquots. To avoid the inactivation of PMSF in <em>aqueous solution,</em> add 5 µl of 200 mM PMSF (in absolute ethanol) per ml of medium just before use (final PMSF concentration 1 mM).</p>
<p>Good results were also obtained with freshwater and terrestrial gastropods using this high osmolarity medium.</p>
<p><strong> </strong></p>
<p><strong>Incubation medium</strong></p>
<p>Phenol - 1.060 mM</p>
<p>4-aminoantipyrine - 0.082 mM</p>
<p>Horseradish peroxidase (» 200 U/mg) - 5 U/ml</p>
<p>Bovine serum albumin - 0.06 % (w/v)</p>
<p>Mannitol (or other polyalcohol) - 25 mM</p>
<p>Potassium phosphate 50 mM pH 7.4</p>
<p>This medium can be stored frozen (-20º C) in aliquots without loss of activity.</p>
<p>In control medium the substrate is omitted.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The following protocol outlines aliquoting resuspended primers, stored at 100µM in stock plates, into working primer plates at a conentration of 5µM for both forward (barcoded) and reverse primers, using the epMotion 5075. The Knigh Lab stores primer combos premixed in working primer plates. 

[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Prepare reagents
?. 
SECTION: Prepare reagents
?. 
SECTION: Prepare Plates
?. 
SECTION: Setup epMotion automation platform.
?. 
SECTION: Prepare Plates
?. 
SECTION: Execute automated protocol
?. 
SECTION: Seal and store plates.
?.