eamag/protocols-with-steps huggingface dataset:
TITLE: Amplification Free Paired End Library Construction Protocol
AUTHORS: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amplification Free Paired End Library Construction Protocol.</div></div>

[STEPS]
?. A total of  of DNA was sheared in a  volume on a Covaris S2 (Covaris, Massachusetts, USA) for 1 cycle of  with a duty cycle of 5%, cycles per burst of 200 and intensity of 3.
600 ng
60 µl
?. The fragmented molecules were then end repaired in  volume using the NEB End Repair Module (NEB, Hitchin, UK) incubating the reaction at  for .
100 µl
22 °C
?. Post incubation  beads of CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were added using a positive displacement pipette to ensure accuracy and the DNA precipitated onto the beads.
58 µl
?. This is then washed twice with 70% ethanol and the end repaired molecules eluted in Nuclease free water (Qiagen, Manchester, UK).
25 µl
?. End repaired molecules were then A tailed in  volume using in the NEB A tailing module (NEB) incubating the reaction at  for .
30 µl
37 °C
?. To the A tailed library molecules  of an appropriate Illumina TruSeq Index adapter (Illumina, San Diego, USA) is added and mixed, then  of Blunt/ TA ligase (NEB) is added and incubated at  for .
1 µl
31 µl
22 °C
?. Post incubation  of stop ligation is added and the reaction incubated at  for .
5 µl
0 Room temperature
?. Following this incubation  beads of CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were added and the DNA precipitated onto the beads.
67 µl
?. The samples are then washed twice with 70% ethanol and the end repaired molecules eluted in  nuclease free water.
100 µl
?. Two further CleanPCR bead based purifications were undertaken to remove any adapter dimer molecules that may have formed during the adapter ligation step. The first with 0.9x volume beads, the second with 0.6x and the final library eluted in  Resuspension Buffer (Illumina).
25 µl
?. Library QC was performed by running a  aliquot on a High Sensitivity BioAnalyser chip (Agilent, Stockport, UK) and the DNA concentration measured using the High Sensitivity Qubit (Thermo Fisher, Cambridge, UK).
1 µl
?. To determine the number of viable library molecules the library was subjected to quantification by the Kappa qPCR Illumina quantification kit (Kapa Biosystems, London, UK) and a test lane run at 10pM on a MiSeq (Illumina) with 2x300bp reads to allow the library to be characterised prior to generation of the 60x coverage required on  the Hiseq2500s (Illumina) with a 2x250bp read metric.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<h4>Method Overview</h4>
<p>This protocol outlines a quick and simple procedure for extracting all organic compounds from a yeast culture for subsequent analysis via Liquid chromatography–mass spectrometry (LC-MS). It is suitable for quickly assessing the presence of products from small sample preps.</p>
<h4>Materials:</h4>
<p>Organic solvents: Ethyl Acetate, Acetone, Acetic Acid and Methanol</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">The purpose of this protocol is to evaluate the significance of delta G values, as a strategy to determine the significance of secondary structure. Sequences are shuffled randomly 100x, maintaining base composition but not base order. The percentile of the delta G vlaue of the non-scrambled (original) sequence is obtained from the distribution created by the delta G values of the 100X scrambled sequences.</span></p>

[STEPS]
?. 
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?.
TITLE: Cryo-EM sample preparation for RCKW:DARPin complex
AUTHORS: Marta Sanz Murillo

[DESCRIPTION]
This is Leschziner's Lab protocol for making cryo-EM grids for RCKW:DARPin complex.

[BEFORE_START]
Decide which protein concentration to use, and create the proper LRRK2 buffers in order to obtain the right salt concentration (150 mM NaCl).

[STEPS]
SECTION: Freezing Grids
1. Plasma clean grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and plasma cleaned them in the Solarus II (Gatan) using the QuantiFoil Au preset.
SECTION: Freezing Grids
3. Apply protein to grids and plunge freeze (3-4 uL)
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze
SECTION: Freezing Grids
4. Store grids in liquid nitrogen until ready for imaging.
SECTION: Freezing Grids
2. Dilute samples to desired concentration in the LRRK2 buffer. Make sure final salt is at 150 mM NaCl.
For best results, make 8 µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.
TITLE: Diagnostic accuracy of pelvic imaging for acute pelvic inflammatory disease: protocol for a systematic review and meta-analysis
AUTHORS: Yuji Okazaki, Yasushi Tsujimoto, Kohei  Yamada, Takashi Ariie, Shunsuke Taito, Masahiro Banno, Yuki Kataoka, Yoshiaki  Tsukizawa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Introduction</span></div><div class = "text-block">Acute pelvic inflammatory disease (PID) is one of the most common causes of the acute abdomen at an emergency department. Women of reproductive age who present acute lower abdominal pain need gynecological examination for the diagnosis of acute PID. It is not well-known how accurate imaging modalities such as contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are for the diagnosis. If these modalities have enough diagnostic accuracy, they can alternate diagnostic tools from the gynecological examination. </div><div class = "text-block"><span style = "font-weight:bold;">Purpose</span></div><div class = "text-block">The purpose of this systematic review is to evaluate the diagnostic accuracy of pelvic imaging for acute PID among women of reproductive age.</div><div class = "text-block"><span style = "font-weight:bold;">Methods and analysis</span></div><div class = "text-block">This systematic review was adhered to the Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies (PRISMA-DTA) statement. We will include all studies on the diagnostic accuracy of pelvic imaging for evaluating acute PID. The target participants are women of reproductive age with acute lower abdominal pain who are suspicious of PID and required gynecological examinations. The target condition is acute PID including any combination of endometritis, salpingitis, tubo-ovarian abscess, and pelvic peritonitis. We expect studies conducted in emergency settings. The index test is pelvic imaging as contrast-enhanced CT and MRI. The reference standard is gynecological examinations defined as examinations by gynecologists using standard diagnostic criteria with or without laparoscopy or transcervical endometrial biopsy. The quality of studies will be evaluated by QUADAS-2. We will present a summary table of evidence, estimates of sensitivity and specificity with 95% confidence interval, and a receiver operating characteristic (ROC). The meta-analysis will be performed using a hierarchical summary ROC model.</div><div class = "text-block"><span style = "font-weight:bold;">Ethnics and dissemination</span></div><div class = "text-block">Data from previously performed studies will be analyzed. We will publish this systematic review in peer-reviewed journals.</div></div>

[STEPS]
TITLE: Dynamin conditional knockout fibroblasts: Tamoxifen inducible Knockout method
AUTHORS: Daehun Park, Pietro De Camilli

[DESCRIPTION]
This cell line was described and characterized in the following paper: Ferguson, S.M.,  Raimondi, A., Paradise, S., Shen, H., Mesaki, K., Ferguson, A., Destaing, O., Ko, G.,  Takasaki, J., Cremona, O., O’Toole, E., De Camilli P. Coordinated actions of actin and  BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Developmental  Cell 17, 811-822, 2009. PMID: 20059951]. This procedure describes tamoxifeninducible KO method using this cell line.

[STEPS]
SECTION: Protocol
1. Culture the cells  at  37 °C and 5% CO2 in DMEM containing 10% FBS,  100 U/ml penicillin, 100 mg/mL streptomycin.

SECTION: Protocol
2. When cells reach 80-90% confluency, detach cells from the dishes using  trypsin-EDTA and split the cells 1:4 and add 2 micromolar (µM) 4-hydroxy-tamoxifen along with  fresh culture medium. Incubate the cells for 2880 min (48 hours). A dish with no 4- hydroxy-tamoxifen is used as a control.

SECTION: Protocol
3. After 2880 min, split the cells once again to prevent cell overcrowding. Add 300 nanomolar (nM)  4-hydroxy-tamoxifen for 4320 min.

SECTION: Protocol
4. Check the depletion of dynamin using immunofluorescence or Western blot.
TITLE: Masson&#39;s Trichrome Staining Protocol for FFPE spheroids and microtissues
AUTHORS: Angela  Denn, Zbigniew  Mikulski

[DESCRIPTION]
The following protocol has been optimized for image analysis-based quantification of collagen within FFPE spheroid samples. It is run on a Leica ST5020 automated stainer. This protocol can be adapted to manual staining.

PURPOSE:
Trichrome stains are used to differentiate collagen from other tissues. They are usually nuclear and cytoplasmic stains, followed by differentiation, then followed by a collagen fiber stain. Trichrome stains are useful for indicating fibrotic changes.

PRINCIPLE:
The staining reaction is based on the differential effect of acid dye on collagen and other tissue components.

[BEFORE_START]
Cut paraffin sections at 4µm.
Slides must be completely dry before deparaffinzation.
Bouin’s solution must be preheated to insure proper mordanting.


[GUIDELINES]
Decreased red staining usually indicates that the staining solution has aged or been overused and should be discarded. If blue staining of connective tissue appears faded, the section has probably been overdifferentiated in the acetic acid solution.
Sections fixed in 10% neutral buffered formalin will stain poorly and unevenly if not mordanted in either Bouin or a mercuric chloride solution; however, mercuric fixatives should not be used because of the toxicity.

[STEPS]
SECTION: Deparaffinization Protocol
1. 
 

SECTION: Mordanting
2. For formalin fixed tissue, post-fix deparaffinized slides in Bouin’s solution for 1 hour at 56°C or at room temperature overnight. Do not leave in oven overnight. Bouin’s solution must be preheated to insure proper mordanting.
Wash in water and discard first two changes to red container aqueous waste. Rinse in sink with running tap water for 5 - 10 minutes to remove the yellow color. Use warm water to remove excess yellow color.
Rinse in sink with distilled water for 30 seconds.
SECTION: Masson Trichrome Protocol MT3B for Leica ST5020 Autostainer
3.  
  StepReagentDurationExactDip1Biebrich Scarlett5:00YY2DI Water station3:00YN3PTA/PMA Working Solution5:00YY4Aniline Blue5:00YN5DI Water station2:00YN6Acetic Acid, 1%2:00YY7IPA, 99%0:20YY8IPA, 99%0:20YY9IPA, 99%1:00YY10IPA, 99%1:00YY11IPA, 99%1:00YY1250:50 Pro-par/IPA991:00YY13Pro-Par3:00NY14Pro-Par3:00NY15Pro-Par   
 
SECTION: Mounting
4. Use a resinous mounting medium to coverslip slides. Allow to dry flat for a minimum of 2 hours.
TITLE: Labelled Building Blocks
AUTHORS: BOC      Sciences

[DESCRIPTION]
Isotope labeled building blocks refer to replacing one or more atoms in building blocks molecular with stable isotopes. Stable isotope labeled building blocks have the advantages of high sensitivity, simple location and accurate quantification. Therefore, stable isotope labeled building blocks are widely used in the field of life science and environment.

Application Fields

Application in Biochemical Field
In the field of biomedicine, stable isotope labeled building blocks could be used to overcome signal degradation during detection of molecule structures by NMR. NMR spectroscopy is an effective means to study the structure and dynamics of building blocks in solution at atomic resolution. However, building blocks analysis by NMR suffers from spectral overlap and line broadening. Isotope labeled building blocks could avoid these questions.
Application in Environment
Isotope labeled building blocks could be used to reveal microbially mediated carbon flow and carbon cycling processes. Isotope-labeled building blocks could indicate the types of carbon sources utilized by microorganisms and the means by which they assimilate these carbon sources, thus providing information on microbially mediated carbon flow and carbon cycling processes, and realizing the relationship between specific microbial communities and the carbon cycle they participate in.

[STEPS]
TITLE: Hematoxylin and Eosin Staining Protocol
AUTHORS: Sierra Simpson, Olivier George

[STEPS]
?. Prepare 95% ETOH in slide staining apparatus Heomtoxylin in slide staining apparatus Eosin in slide staining apparatus
?. Stain in Mayer's Hematoxylin
?. Rinse slides 3X 15 min in tap water - if tap water is acidic, prepare Scott's tap water recipe in sigma insert
?. Rinse with DI water 30 Seconds
?. Place slide in 95% Ethanol for 1min
?. Place in Eosin Y for 1 min
?. Dehydrate and clear through 2 changes each 95% Ethanol 2 minutes 100% Ethanol 2 minutes Xylene ( Histoclear as a substitute ) EMS Cat# 64114
?. Mount with resinous mounting medium 200ul / slide.
TITLE: Transcardiac Perfusion of Mouse for Brain Tissue
AUTHORS: Maryana Nissan, divya.darwinarulseeli

[DESCRIPTION]
Transcardiac perfusion is a method used for clearing blood and preserving the mouse brain for immunostaining. 


[STEPS]
1. Add Neutralizing Agent to break down Paraformaldehyde [PFA] for proper disposal. We use Hydeaway [CAT #2201 from Decon Labs] 
2. Turn on perfusion apparatus and place tube into 1X Phosphate-Buffered Saline [PBS] 
3. Wash system for 1 round, until the tube is saturated with 1X PBS 
4. Saturate anesthetic chamber with Isoflurane
5. Place mouse in anesthetic chamber for about 1 minute 
6. Remove mouse from anesthetic chamber and check reflexes by pinching foot
7. If reflex is active, place mouse back into chamber. If not, proceed
8. Grip the skin on the chest with forceps, make an incision beneath the ribcage to expose the diaphragm and liver 
9. Carefully make incisions along the diaphragm to expose the heart
10. Tighten the skin with hemostatic forceps, twist, and retract back to expose the heart 
11. Secure the heart with forceps with minimal pressure, insert needle about a depth of 1.5 mm into the left ventricle 
12. Puncture the aorta, causing dark venous blood to flow out immediately 
13. Wait until the blood gets cleared and lighter in color, until you notice white drops 
14. Switch out the tube from 1X PBS into 4% PFA 
15. 4% PFA will cause the body to become stiff 
16. Allow 100mL of 4% PFA to circulate the body
17. Stop circulation and remove needle from the heart
TITLE: Embedding organoids in agarose 
AUTHORS: Gabriela Vallejo Flores, Annika Fendler

[DESCRIPTION]
This protocol is for embedding organoids in low melting point agarose, the protocol was adapted from Fendler, A., et al. Research Square ( 2020).

[BEFORE_START]
Before starting the protocol,  prepare the following solutions and material:

0.1% BSA in PBS(w/v), 
10% NBF in PBS(v/v) 
1% BSA  in PBS. 
Work with 1% BSA/PBS coated tubes and pipetts
Precool centrifuge
Prepare the 80°C heating block the day of the agarose embedding 


[GUIDELINES]

Before starting the protocol,  prepare the following solutions  1% BSA in PBS(w/v),  0.1% BSA in PBS(w/v), 10% NBF in PBS(v/v) 
This protocol include  one overningt incubation


[STEPS]
SECTION: Organoids embedding in agarose
2. Remove the supernatan from the well and add 1 mL   to release the organoids from the matrigel, 500 x g, 4 min, 4 °C

SECTION: Organoids embedding in agarose
3. Wash the organoids with              1 mL , and  300 x g, 4 min, 4 °C and discard the supernatant.


SECTION: Organoids embedding in agarose
4. Resuspend the organoids in1 mL and fix over night at 4°C rocking.
SECTION: Organoids embedding in agarose
5. Wash the organoids  with              1 mL , and  300 x g, 5 min, 4 °C and discard the supernatant.
resuspend in  1 mL
SECTION: Organoids embedding in agarose
10. After the agarose has polymerized, release the agarose bead from the Eppendorf tube, Use a razor blade to cut off the bottom of the Eppendorf tube at the opposite side as the organoid pellet, and carefully push out the agarose bead. 
SECTION: Organoids embedding in agarose
11. Trim the bead around the organoid pellet, transfer it to an embedding cassette, and place the cassette in 70 % volume     

SECTION: Organoids embedding in agarose
6. PAUSE POINT: The Organois can remain in 0.1% of BSA for a month. 
SECTION: Organoids embedding in agarose
7.           300 x g, 4 min, 4 °C and discard the supernatant.
SECTION: Organoids embedding in agarose
1. Sample description, Table 1.

 

SECTION: Organoids embedding in agarose
8. Shortly place the tube in  an 80 °C before carefully overlaying the organoids pellet with 400 µL .

SECTION: Organoids embedding in agarose
9. 300 x g, 4 °C let the agarose solidify on ice.

SECTION: Organoids embedding in agarose
12.  The agarose bead can remain in 70% EtOH for up to 7 days before dehydration. 
SECTION: Organoids embedding in agarose
13. Next step go to the following protocol: Organoids dehydratation and Organoids embedding in paraffin
SECTION: Organoids embedding in agarose
14.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Step-by-step description on the use of iPATH3 on Uproc-DNA outputs.

Path3.0 (http://pathways.embl.de) is a web-application for the  visualization and analysis of cellular pathways. It is freely available  and open to everyone. Currently it is based on four KEGG global maps,  which summarize up to 158 traditional KEGG pathway maps, 192 KEGG  modules and other metabolic elements into one connected and manually  curated metabolic network. Users can fully customize these networks and  interactively explore them through its redesigned, fast and lightweight  interface, which highlights general metabolic trends in multi-omics  data. It also offers navigation at various levels of details to help  users further investigate those trends and ultimately uncover novel  biological insights. 

More information about iPATH3 can be found here : https://academic.oup.com/nar/article/46/W1/W510/4990021

[BEFORE_START]
This protocol requires you to have a "counts" output file from Uproc. To learn more on Uproc and how to run this tool using the iMicrobe platform, read this protocol.


[STEPS]
SECTION: Create a pathway map for a unique sample
?. 
SECTION: Create a pathway map comparing two samples
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Anti-BrdU Staining Protocol using DNAse with surface and fluorescent proteins</p>

[STEPS]
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TITLE: Online Survey Among Pregnant Women With Medical Indication Or Wish For Elective Caesarean Section On Second Opinions In Germany: Study Protocol
AUTHORS: Nadja Könsgen, Anke Kaulbert, Barbara Prediger

[DESCRIPTION]
Background: Caesarean section (CS) rates have been continuously rising worldwide over the last decade without being accompanied by significant health benefits. Various approaches are discussed internationally to limit the proportion of caesarean deliveries to the necessary cases. The proposed study aims to assess the perspective of pregnant women with medical indication or wish for elective CS on second opinions The goal is to examine whether they consider a second opinion useful to facilitate their decision-making and/or certainty for the mode of delivery, and under which circumstances they would seek a second opinion. 
Methods: The study population will comprise adult pregnant women (18 years or older) with medical indication or wish for elective CS (both currently pregnant women and persons for whom the indication was given in a past pregnancy). The survey will be conducted online and the data will be evaluated using quantitative descriptive analysis.
Discussion: The survey is an opportunity to get a comprehensive picture from the perspective of pregnant women. In combination with our survey of health professionals it paves the way for following research in this area.The results might be limited by a possible selection bias of the study population. 

[STEPS]
TITLE: RT-LAMP SARS-CoV-2 positive control stock
AUTHORS: Christina Newman, Andrea Weiler, Dawn Dudley, Mitchell Ramuta, Amelia Haj, Shelby O'Connor, David O'Connor

[STEPS]
?. Using either the company-provided stock concentration or a qRT-PCR determined stock concentration, dilute virus in dH2O to create a 1e9 copies/ml stock virus.
?. Create a 1e6 copies/ml stock virus by diluting 1e9 copies/ml stock 1:1000 in 50% dH2O and 50% 1x PBS solution.
?. Create a ready-to-use positive control stock (1e3 copies per 3 µl) for RT-LAMP saliva testing assay by diluting 100 µl of the 1e6 copies/ml stock in 200 µl of 50% dH2O/50% 1x PBS solution.
?. Prepare 10 µl aliquots of ready-to-use positive control stock in PCR strip tubes.
TITLE: Salmonella live attenuated vaccine preparation.
AUTHORS: Angel  Justiz-Vaillant, Suzette Curtello

[STEPS]
?. A Salmonella cocktail (mixture of five Salmonella strains: S. Montevideo, S. Yeerongpilly, S. Augustenborg, S. Kentucky and S.Typhimurium) is incubated at 37°C in peptone broth with combinations of natural extracts.
?. Preparation includes clean, sun dried onion and garlic cloves.
?. After a week of drying, onion and the cloves of garlic is separately grounded using electric blender,
?. Dilute the plant preparations in 1L distilled water.
?. Each extract is used individually and in combined concentration.
?. Five ml of the diluted extract (15 mg/ml) is incorporated in differential and selective Salmonella plating media.
?. A bacteriogram is carried out to check for bacteria attenuation.
?. The immunogen  contains 5 serovars of Salmonella (10^8 CFU of vaccine strain) per chicken in 1 ml of incomplete Freund’s adjuvant.
?. It is given intramuscularly at chicken breast
?. The immunogen (5 serovars) without incomplete Freund’s adjuvant (IFA) is given orally (10^8 CFU per chicken).
?. All Salmonella isolates were investigated for their antibiotic resistance with the disc diffusion test using the following discs (Difco):
?. gentamicin (10 μg), kanamycin (30 μg), ampicillin (10 μg), amikacin (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), chloramphenicol (30 μg), cefazolin (30 μg), cephalothin (30 μg), cefepime (30 μg), cefotaxime (30 μg), streptomycin (10 μg), cefoxitin (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), tetracycline (30 μg ) and imipenem (10 μg).
?. The five Salmonella serovars were fully attenuated and inoculated in appropriate media and store at -20 degree Celsius.