eamag/protocols-with-steps huggingface dataset:
TITLE: Comparative Ct method quantification (2-ΔCt method).
AUTHORS: Barbara Rizzacasa, Elena Morini, Ruggiero Mango, Chiara Vancheri, Simone Budassi, Gianluca Massaro, Sara Maletta, Massimiliano Macrini, Silvio D’Annibale, Francesco Romeo, Giuseppe Novelli, Francesca Amati

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For the evaluation of the expression level of each miRNA was used the Ct (threshold cycle) method quantification. The threshold cycle (Ct) is defined as the PCR cycle at which the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold. This method allows to quantify  the absolute expression of each miRNAs in each sample analyzed and then to calculate the different expression of each miRNA in sample versus the controls.</div></div>

[STEPS]
?. [Evaluation of the absolute expression of miRNAs in each sample analyzed:]
For each sample is obtained an amplification curve in which the Ct is inversely proportional to the initial sample quantity;
?. [Evaluation of the absolute expression of miRNAs in each sample analyzed:]
Normalize the data to an internal control and calculate DCt = (Ct gene of interest - Ct internal control);
?. [Evaluation of the absolute expression of miRNAs in each sample analyzed:]
Calculate 2-ΔCt   value that is the measure of the microRNA expression level in each sample analyzed.
?. [Evaluation of the expression of each miRNA in two different samples (2-ΔΔCt  value):]
Calculate DDCt= [(Ct gene of interest-Ct internal control) sample A] – [(Ct gene of interest-Ct internal control) sample B];
?. [Evaluation of the expression of each miRNA in two different samples (2-ΔΔCt  value):]
Calculate 2-ΔΔCt   value that is the measure of the microRNA expression in each sample analyzed compared with control sample.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes fluorescence-activated single cell sorting using a MoFlo Astrios EQ cell sorter (Beckman Coulter) and whole genome amplification of single sorted cells in plate formats using the REPLI-g Single Cell kit (Qiagen).</p>

[GUIDELINES]
<p><strong><em>General cleanroom rules<br /></em></strong></p>
<p>FACS sorting and whole genome amplification is performed in a cleanroom. Protective clothing such as lab coats, shoe covers, are required for entering the room and additional protection with gloves, hair nets and mouth covers are needed when the plates are sorted into and further processed. Lab coats should be dedicated to a specific task/analysis i.e. sorting or whole genome amplification. The screening of the amplified material is done outside the cleanroom in a PCR hood dedicated to DNA work. To keep a clean environment, take off your gloves when using the computer or touch screen. For a more detailed description of the different steps and critical issues see the paper by Rinke et al 2014 (1).</p>
<p> </p>
<p><strong>Whole genome amplification</strong></p>
<p>Whole genome amplification is performed as described for the “amplification of genomic DNA from single cells” in the REPLI-g Single Cell Handbook (2), with the exception that volumes are scaled down with a quarter of the original volume, hence 12.5 µl in total.</p>
<p> </p>
<p>Lysis and neutralization reagents from the REPLI-g Single Cell kit are first added to each well of the sorted plate to facilitate genome amplification. The MDA master mix is then added, the plate is sealed with an optical sealing and the reaction is monitored every 15 minutes in real-time at 30 °C for 8 h using a DNA-binding dye (SYTO13) and an instrument suitable for fluorescent readings e.g. a PCR machine or a plate reader. Note that the REPLI-g Single Cell kit mastermix should not be UV-decontaminated as it is already treated to minimize contamination. Tubes, water, lysis and stop solution can however be UVed for 2 Joules before use.</p>

[STEPS]
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TITLE: Time of flight mass cytometry (CyTOF)
AUTHORS: Peter Hinderlie, Philippa R Kennedy

[DESCRIPTION]
Staining procedure for cells analyzed by time of flight mass cytometry.

[STEPS]
3. Cells are then fixed using 2% PFA, for 20mins.

4. For intracellular staining, cells are permeabilized by incubation with Triton X 0.1% for 5 min at room temperature, followed by incubation with intracellular antibody cocktail for 30 min at 4 °C.

1. Cells from separate donors or conditions are stained separately with Cisplatin (Cat. No. 201064, Fluidigm) followed by barcoding using the Cell-ID 20-Plex Pd Barcoding Kit (Cat. No. 201060, Fluidigm). 
2. Following barcoding, samples are combined into a single 5 mL polystyrene U-bottom tube and incubated in the surface marker antibody cocktail for 30 min at 4 °C
5. Stained cells are incubated overnight with Cell-ID Intercalator (Cat. No. 201192A, Fluidigm).
6. The following morning, cells are washed in either Maxpar PBS (Cat. No. 201058, Fluidigm), Maxpar Cell Staining Buffer (Cat. No. 201068, Fluidigm), or Millipure Water, with a spin at 1600 RPM for 4 min to remove the supernatant and replace it with fresh buffer.
7. Samples are run on a time of flight mass cytometer (CyTOF 2, Fluidigm) and analyzed with Cytobank (Cytobank Inc., RRID:SCR_014043) and Astrolabe Diagnostics (https://astrolabediagnostics.com/)
TITLE: LC3-lipidation-assay
AUTHORS: Liv Jensen

[DESCRIPTION]
Protocol for an in vitro LC3 lipidation assay using purified proteins and synthetic liposomes.

[STEPS]
1. Mix 2μM ATG3, 2μM ATG7, 1μM WIPI2, 200nM ATG12–ATG5-ATG16L1-GFP, 5μM LC3B
1:1 with extruded liposomes in reaction buffer.
2. Incubate at 37 ̊C
3. At 0, 15, 30, 60, and 120 minutes, remove 12μl of reaction mixture andquench by
adding 4ul of SDS-PAGE loading bufferandheatingat 60 ̊C for 10 minutes.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Nucleic acid precipitation is used to concentrate and/or purify nucleic acids. The below protocol is based on the fact that nucleic acids are less soluble in alcohol than in more polar water. Addition of salt further decreases solubility by competing for water dipoles; as does low temperature. Please see the <a href="http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids" target="_blank">OpenWetWare</a> website for more details.<br /><br /><strong>Citation</strong>: OpenWetWare contributors, 'Ethanol precipitation of nucleic acids',&nbsp;<em>OpenWetWare, ,</em>&nbsp;1 July 2012, 10:48 UTC, &lt;<a class="external free" title="http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420" href="http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420" target="_blank">http://openwetware.org/index.php?title=Ethanol_precipitation_of_nucleic_acids&amp;oldid=611420</a>&gt; [accessed 21 January 2015]

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to make an acoustic droplet ejection compatible plate for liquid handling on the Echo 555 using an epMotion 5075. The protocol expects (4) 96-well gDNA plates and an empty 384-well polypropylene (PP) echo destination plate. The 4 plates will be compressed in a fully interweaved layout. The following images depict this pattern. 
  

 





[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 gDNA plates
?. 
SECTION: Prepare gDNA plates
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SECTION: Setup epMotion automation platform
?. 
SECTION: Execute automated protocol
?.
TITLE: Isolation of nuclei from frozen human skeletal and cardiac muscle for single nucleus RNA and chromatin assays 
AUTHORS: James  R Hocker, Sebastian  Preissl, Dinh H Diep

[DESCRIPTION]
This protocol was adapted for the isolation of single nuclei from frozen skeletal and cardiac muscle tissues for molecular characterization with the SNARE-seq2, sci-ATAC-seq, and snRNA-seq assays.




References:
(1) Preissl et al (2015). Circulation Research. Doi: 10.1161/CIRCRESAHA.115.306337
(2) Hocker et al (2021). Science Advances. Doi: 10.1126/sciadv.abf1444

[STEPS]
1. Section flash-frozen skeletal muscle into aliquots according to desired nuclei yield and store on dry ice or at -80 °C .


 

2. Prepare buffers and chill components:

 
Prepare buffer fresh on day of nuclei isolation
Precool centrifuge to 4 °C 
Precool all buffers at 4 °C 
Place gentleMACS dissociator in cold room / chiller
Chill gentleMACS M tubes on ice
Chill 5.0 mL Eppendorf tubes on ice

3. Transfer sectioned frozen tissue to gentleMACS M tubes.    
4. Add the recommended volume of MACS buffer to gentleMACS M tube. 
 

5. Homogenize with Miltenyi tissue dissociation protocol: "Protein_01_01" on the gentleMACS instrument in the chiller/cold room.  
6. Briefly centrifuge the gentleMACS M tube to pull all the homogenate to the bottom.160 x g, 15 s, 4 °C 

7. Filter homogenate through 30 uM CellTrics filter into 5 mL Eppendorf Tubes.

8. Wash gentleMACS M tube with another 1 mL of MACS buffer and filter the wash.
9. 900 x g, 10 min, 4 °C 
 


10. Decant and discard the supernatant.
11. Resuspend the pellet with the recommended NPB buffer.
12. Gently rotate the sample in cold room/chiller for 10 min .

 

13. Centrifuge permeabilized nuclei at 900 x g, 10 min, 4 °C .

14. Decant and discard the supernatant.
15. Resuspend the pellet in 500 uL PBS + RNase In.

 

16. QA/QC: Count the nuclei. Mix 1:1 volume of cellular suspension with a staining solution (ie DAPI). Load 10 uL of mixture onto a Biorad Cell Counter slide then count with a BioRad TC20 Cell Counter. Gate 4 uM-6 uM for nuclei sizes. 

17. QA/QC: Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the basic steps neccessary to maintain and propagate Aiptasia as they are done in the Pringle lab.</p>

[GUIDELINES]
<p>If working with symbiotic anemones hosting different <em>Symbiodinium</em> strains make sure to kepp all working surfaces clean and wash/sanitize your hands regularly.</p>
<p>Keep different strain combinations as separated as possible (e.g. different shelves or incubator compartments). Do water changes and tank cleaning for one host-symbiont combination at a time. Sanitize hands and surfaces after finishing one batch of host-symbiont combination.</p>
<p> </p>
<p>Aiptasia is a very robust organism. Especially the symbiotic animals are more sensitive to impaired water quality then to short-term starvation (1-2 weeks).</p>
<p> </p>
<p>The bigger the anemones the more feeding they need to keep their body mass.</p>

[STEPS]
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TITLE: Anaesthesia of Lumpfish (Cyclopterus lumpus) fries.
AUTHORS: Julianne Valla Jacobsen, Kjell J. Nilssen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Large numbers of lumpfish are produced for the Norwegian salmon industry and are used to combat sea lice infestations. The present work tested the efficacy of three anaesthetic chemicals on lumpfish fries (average weight of 0.97 g).</div></div>

[STEPS]
?. [Prepare anaesthetic bath for immersion anaesthesia.]
The following chemicals and concentrations were used in our study:Liquid Benzoak was stirred into seawater (12.5, 25, 37.5, 50, 100 mg/L), metacaine powder and the corresponding weight of sodium bicarbonate (Na2CO3) were dissolved in seawater at concentrations of 25, 38, 44, 60, 75, 100, and 150 mg/L. Isoeugenol (540 mg/ml) was diluted in ~5 mL heated (30oC) water prior to being added to the seawater to yield 6, 12.5, 18, 25, and 50 mg/L.
?. [Immersion anaesthesia.]
Transfer individual lumpfish fries from the holding tank into an aerated transparent box (5L) containing pre-mixed chemicals.
?. [Immersion anaesthesia.]
The time to induce stage 3b was measured for 10 lumpfish. The fish were considered to be surgically anaesthetized (known as stage 3b) at the last behavioural stage (immobile and weak respiration).  Observations were terminated if the anaesthesia was not reached within 10 min (600 sec), while the chemical concentrations inducing stage 3b within ~3 to 5 min were regarded as the Selected Best (SB) concentrations.
?. [Recovery - therapeutic window.]
To investigate the safety margin of the anaesthetics (therapeutic window) batches of 10 lumpfish were exposed to the SB anaesthetic concentration for 5, 10, and 20 min.
?. [Recovery - therapeutic window.]
Thereafter, the fries were transferred to individual containers (~1 L) holding only seawater.
?. [Recovery - therapeutic window.]
The time to full recovery (the point at which they managed to swim normally) was measured. Observations were terminated (T) if the fish had not recovered within 15 min (900 sec).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for running an illumina Hiseq4000 sequencer, following the process from the loaded flow cells through the sequencing run. The HiSeq 4000 uses a dual flow cell capable of processing over 400 Gb per day or 1.5 Tb per run, with up to 5 billion single reads. See other protocols for DNA extraction or library construction and loading of flow cells.</p>

[GUIDELINES]
<p>See the illumina SOP here: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/hiseq4000/hiseq-4000-system-guide-15066496-05.pdf</p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 53">
<div>
<div>
<p>TG in plasma was measured with the calibrated automated thrombogram (CAT) assay as developed by Hemker and co-workers [18-20]. Briefly, 80 μl platelet poor plasma (PPP) was mixed with 20 μl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 μM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 μl of calibrator (α2macroglobulin- thrombin complex, [19]) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z- Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 μM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) [20]. Thrombin generation was expressed based on endogenous thrombin potential (ETP); lagtime (LT); thrombin peak (TP), time-to-thrombin peak (TTP).</p>
</div>
</div>
</div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background:</strong> Acute lung injury is a life threatening condition often requiring mechanical ventilation. Lung-protective ventilation with tidal volumes of 6 mL/kg predicted body weight (PBW, based on sex and body height), is part of current recommended ventilation strategy. However, it is a common practice to visually estimate the body height of mechanically ventilated patients and use these estimates for ventilator settings. We aimed to determine if the common practice of estimating visual height to define tidal volume reduces the possibility of receiving lung-protective ventilation.</p>
<p> </p>
<p><strong>Methods:</strong> In this prospective observational study, 28 mechanically ventilated patients had their height visually estimated by 20 nurses and 20 physicians. All medical professionals calculated the PBW and a corresponding tidal volume with 6 ml/kg/PBW on the basis of their visual estimation. The patients’ true heights were measured and the true PBW with a corresponding tidal volume was calculated. Finally, estimates and measurements were compared. </p>
<p> </p>
<p><strong>Results:</strong> 1033 estimations were undertaken by 153 medical professionals. Most estimates were imprecise and resulting data comprised taller body heights, higher PBW and higher tidal volumes (all p≤0.01). Using estimates for tidal-volume definition, patients are exposed to mean tidal volumes of 6.5 ± 0.4 ml/kg/PBW. 526 estimation-based tidal volumes (51.1%) did not provide lung-protective ventilation.</p>
<p> </p>
<p><strong>Conclusion:</strong> The common practice of visually estimating body height and using these estimates for ventilator settings is imprecise and potentially harmful because it reduces the chance of receiving lung-protective ventilation. Avoiding this practice increases the patient safety. Instead, height should be measured as a standard procedure.</p>
<p> </p>

[STEPS]
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TITLE: Coverslipping: Use of the Leica CV5030 Coverslipper
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the coverslipping of glass slides with the Leica CV5030.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: SOP52v1_TGD_IIDP-HIGISampleProcessing_Public
AUTHORS: Varsha Rajesh

[DESCRIPTION]
This protocol details the workflow to process pancreatic tissues (received from the Integrated Islet Distribution Program's distribution centers) for genotyping at the Translational Genomics of Diabetes Lab at Stanford University.

[STEPS]
SECTION: Receiving Samples
1. When acinar samples are received from the various isolation centers, match what is physically sent with sample info list sent with package and make sure all samples are accounted for. Take note of the condition of the samples. 
SECTION: Receiving Samples
2. Let isolation center know samples were received and in what condition. 
SECTION: Receiving Samples
3. Print QR barcodes for the acinar samples in this format and tape onto the tubes. 

HIGI#ACIN (# being the next sequential number)
SECTION: Receiving Samples
4. Store tubes in Yalow -80°C freezer in  "IIDP - HIGI Acinar Samples" box.
SECTION: Extracting DNA
5. Thaw acinar tubes on ice. 
SECTION: Extracting DNA
6. Use DNA extraction protocol for DNeasy Blood & Tissue Kit (https://protocols.io/view/dna-extraction-qiagen-dneasy-blood-amp-tissue-kit-bjngkmbw). 
SECTION: QC
7. After extraction, measure the concentration by nanodrop, taking account of A260/280 and A260/230 values as well. A260/280 value should be close to 1.8 and A260/230 should be around 1.8-2.0. 
SECTION: QC
8. Take a portion of the sample to measure concentration by Qubit. Protocol can be found here: https://protocols.io/view/qubit-dsdna-assays-bnstmeen. For genotyping, Qubit concentration should be above 50 ng/uL and total DNA should be above 750 ng. 
SECTION: Genotyping
10. Record sample info, nanodrop QC and Qubit information in genotyping QC spreadsheet, which will be sent to the SFGF core whenever the entire genotyping batch is ready (follow this protocol: https://www.protocols.io/edit/sop54v1-tgd-genotypingsubmissionforcollaborators-b8jaruie).
SECTION: QC
9. Create QR codes that include sample name in this format: HIGI#DNA. Label should also include nanodrop concentration and QC info. 
SECTION: Genotyping
11. As of 2023-12-01, samples were sent to be genotyped on Illumina's Infinium Omni Exome 2.5 arrays.
As of 2024-10-31, samples were sent to be genotyped on Illumina's Infinium Global Diversity Array-8+ v1.0   Omni2.5-Exome-8v1.6 arrays.
TITLE: Human Embryonic Kidney Cells (HEK-293)
AUTHORS: Md Razaul Karim

[DESCRIPTION]
This protocol details the Human Embryonic Kidney Cells (HEK-293) culture.

[STEPS]
SECTION: Day-01 (Mon)
1. Plating with DMEM full media .
SECTION: Day-01 (Mon)
2. Warm DMEM full media, PBS, and Trypsin in the 37 °C bead bath for 30 min. Clean the working area by using 70% ethanol.
SECTION: Day-01 (Mon)
2.1. Sup out old media without touching cells.
SECTION: Day-01 (Mon)
2.2. Wash by adding 5 mL PBS slowly, rinse, and rock back and forth.
SECTION: Day-01 (Mon)
2.3. Add 2 mL-3 mL trypsin (0.25%); keep in incubator for 3 min.
SECTION: Day-01 (Mon)
2.4. Check under microscope if cells are detached, add 5 mL media and transfer to a tube.
SECTION: Day-01 (Mon)
2.5. Spin   for 3 min.
SECTION: Day-01 (Mon)
2.6. Sup out and add 10 mL fresh media & re-suspend cells gently and carefully.
SECTION: Day-01 (Mon)
2.7. Count cells density and split accordingly. 15,000 cells/ml for maintenance.

(i) Usually 1.0-1.5x10^4/ml cells for Biochem, and 
(ii) 0.5x10^4/ml cells for IF.
SECTION: Day-02 (Tue)
3. Rest.
SECTION: Day-03 (Wed)
4. Replace with DMEM full media/Drug treat.
SECTION: Day-04 (Thu)
5. Drug treat if necessary /Harvesting.
SECTION: Day-05 (Fri)
6. Drug treat if necessary /Harvesting.
TITLE: Aliquotting PCR Primer Mixture
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to take lyophilized PCR primers and dilute and aliquot into useful quantities and concentrations.</div></div>

[STEPS]
?. [Dilute primers to 100 μM]
Add the volume of nuclease-free water to each tube according to the documentation provided by the primer.In the case of E. coli K12, the following volumes are to be used:  AB1OligoVolume2F184031552 μL3R18551728 μL
AB1OligoVolume2F184031552 μL3R18551728 μL
?. [Make 100 μL stock ]
Use the following table to make 25 μM, 100 μL stocks:  AB1ComponentVolume2100 μM F18403125 μL3100 μM R1855125 μL4Nuclease Free Water50 μL5Total100 μL
AB1ComponentVolume2100 μM F18403125 μL3100 μM R1855125 μL4Nuclease Free Water50 μL5Total100 μL
?. [Make 100 μL stock ]
Store all primer mixtures in freezer for up to 2 years
?. [Aliquot primer mixtures ]
Aliquot the primer mixture into quantities that can be expected to be used in one day to avoid freeze/thaw cycling.