eamag/protocols-with-steps huggingface dataset:
TITLE: High throughput Zika virus reporter virus particle microneutralization assay: Laboratory Procedure
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Zika virus is a Flavivirus, transmitted via </span><span style = "font-style:italic;">Aedes </span><span>mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate-based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (</span><span style = "font-style:italic;">Renilla </span><span>luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon </span><span style = "font-style:italic;">Renilla</span><span> reporter construct. </span></div><div class = "text-block"><span>Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed </span><span style = "font-style:italic;">Renilla </span><span>luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. </span></div><div class = "text-block"><span>RVP EC</span><span style = "vertical-align:sub;">50</span><span> titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R</span><span style = "vertical-align:super;">2</span><span>>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the library preparation for Nanopore sequencing according to the SQK-MAP006 protocol. </p>
<p>It accompanies the <em>GigaScience</em> publication:</p>
<p> </p>
<p>Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. <em>GigaScience...</em></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: RNA Extraction Protocol for Shorea
AUTHORS: mpfsum 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>RNA extraction protocol using CTAB method optimized for leaf and bud samples from </span><span style = "font-style:italic;">Shorea </span><span>sp.</span></div></div>

[STEPS]
?. [Lysis]
Weigh BUD  or LEAF  and transfer into a 1.5 mL tube.
[(25-40 mg)]
[(50-70 mg)]
?. [Lysis]
Incubate at  for .
60 °C
?. [Resuspension]
Resuspend the pellet in  RNAse-free water.Flick the tube until the pellet is no longer visible.If the pellet is difficult to dissolve, leave it for ~30 min before flicking again.If it still does not dissolve, add more RNAse-free water AND adjust the subsequent steps accordingly.
50 µl
?. [Genomic DNA Digestion]
Removed gDNA using .Follow the manufacturer's protocol.
?. [Purification]
Purification is done using with minor adjustment to the protocol.Add  RLT buffer.Mix gently (flicking).
350 µl
?. [Quality Assessment]
Evaluate the RNA quality using Agilent 2100 Bioanalyzer.
?. [Lysis]
Ground in CTAB buffer (3% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl pH 8.0, 0.2% b-mercaptoethanol) using tissuelyzer.
?. [Precipitation]
Add 1 volume of chloroform.Mix by vortex but not too vigorous.Ensure that the two phases of the mixture are homogenized well.
?. [Precipitation]
Centrifuge at .Transfer the supernatant into a new 1.5 mL tube.
Centrifuge: 5000 33, 1 min
?. [Precipitation]
Add 2/3 volume of isopropanol.Mix gently (turning the tubes upside down  ).
?. [Precipitation]
Centrifuge Discard solution carefully.
Centrifuge: 5000 33, 5 min
Formation of white to translucent pellet.
?. [Washing]
Add  ethanol.Mix gently (flicking the tube) until the pellet no longer stick to the tube.
700 µl
?. [Washing]
Centrifuge .Discard solution carefully.
Centrifuge: 5000 33, 5 min
?. [Washing]
Air dry the pellet .Repeat Washing if necessary.
?. [Purification]
Add  absolute ethanol.Mix gently (flicking).
250 µl
?. [Purification]
Transfer the solution into RNeasy Mini Spin column.Centrifuge .Discard the flow through.
Centrifuge: 10000 33, 15 s
?. [Purification]
Add  RW1 buffer to the column.Centrifuge .Discard the flow through.
700 µl
Centrifuge: 10000 33, 15 s
?. [Purification]
Add  RPE buffer to the column.Centrifuge .Discard the flow through.
500 µl
Centrifuge: 10000 33, 15 s
?. [Purification]
Add  RPE buffer to the column.Centrifuge .Discard the flow through.
500 µl
Centrifuge: 10000 33, 2 min
?. [Purification]
Replace the collection tube.Centrifuge  to further dry the membrane.
Centrifuge: 14000 33, 1 min
?. [Purification]
Replace the collection tube with a new 1.5 mL tube.Add  RNAse-free water directly onto the membrane.Incubate at  for .
50 µl
0 Room temperature
?. [Purification]
Centrifuge .Store the extracted RNA in  or  for longer storage.
Centrifuge: 10000 33, 1 min
-20 °C
-80 °C
TITLE: Restriction endonuclease reactions
AUTHORS: Shuning    Guo

[DESCRIPTION]
This protocol is used to having restriction endonuclease reactions based on endonuclease by NEB.

[STEPS]
1. Add the following reagents to a PCR tube (e.g. 50μl) on ice.

 

2. Incubate at 37℃ in water bath for 1 hour.
3. Stop the reaction by heat inactivation. Temperature and time for inactivation depends on the enzyme.
4. Use electrophoresis to confirm if correct construct was present.
TITLE: CAMbank: CPT Field Processing v1
AUTHORS: Eliah G Overbey

[DESCRIPTION]
Field processing of CPT vacutainers for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: Plasma, PBMCs, and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes 8 to 10 times to fully mix in the sodium citrate anticoagulant.

Store the tube upright at room temperature until centrifugation.

To ensure proper barrier formation, blood samples should be centrifuged within 2 hours of blood collection. Centrifugation more than 2 hours after specimen collection may cause incomplete barrier formation.
SECTION: Centrifuge Settings
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 0
temperature: RT
duration: 30 minutes
speed: 1800xg
SECTION: Centrifuge Settings
3. Place the CPTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube breakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Aliquot Plasma
4. Carefully remove CPTs from the centrifuge and inspect for separation of red blood vs PMBCs vs plasma layers. 

Transfer CPTs to a sanitized laminar flow hood. 
SECTION: Wash PBMCs
6. Prepare the wash buffer (PBS + 2% FBS)

In a 50mL conical tube, mix:
24.4mL of PBS
600µL of FBS

Create more wash buffer as needed (depending on the number of tubes drawn). 
SECTION: Wash PBMCs
7. Add 5mL PBS + 2% FBS to each CPT tube.

Gently resuspend the peripheral blood mononucleocyte cells (PBMC) layer into the PBS + 2% FBS using a serological pipet.
SECTION: Aliquot Plasma
5. Using a P1000, carefully aliquot plasma (yellow layer above buffy layer) into 2D barcoded tubes at 500µL each. 

Do not pipet close enough to disturb the buffy coat (the cloudy PBMC cell layer).

Return 2D tubes with plasma to the rack and place in the -80C freezer.
SECTION: Wash PBMCs
8. One CPT tube at a time, transfer the resuspended cells into a new 15mL conical tube.
SECTION: Wash PBMCs
9. Add fresh wash buffer to bring the conical tube volume to 15mL. 
SECTION: Wash PBMCs
10. Repeat steps  8-9 for all CPTs.
SECTION: Wash PBMCs
11. Pellet the PBMCs in the centrifuge. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 15 minutes
speed: 300xg


SECTION: Wash PBMCs
12. Return the 15mL conical tubes to the laminar flow hood. 
Gently aspirate the media away from the cell pellet. 
SECTION: Viably Freeze Cells
13. Prepare freezing media: 10% DMSO, 90% FBS. 
One CPT will require 6mL of freezing media.

SECTION: Viably Freeze Cells
14. Resuspend the cells of each 15mL conical tube in 6mL of freezing media. 
SECTION: Viably Freeze Cells
15. Transfer 1mL of PBMCs into each cryovial. 
SECTION: Viably Freeze Cells
16. Place the cryovials into Mr. Frosty containers.
SECTION: Viably Freeze Cells
17. Store the Mr. Frosty's at -80C overnight, then ship back to WCM for liquid nitrogen storage. 
18. Recap and freeze the CPT tubes to preserve DNA in the red blood cell pellet
TITLE:  UC Davis - Intravenous Glucose Tolerance Test
AUTHORS: Kent Lloyd, Kristin Grimsrud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>The intravenous glucose tolerance test is used to assess insulin sensitivity, glucose disposal and islet function </span><span style = "font-style:italic;">in vivo</span><span> by measuring glucose and insulin responses following intravenous glucose administration.</span></div></div>

[STEPS]
?. Intravenous Glucose Tolerance Test (IVGTT):1. Fast mice prior to IVGTT evaluation.  The fasting duration may vary depending on the investigators preference but may not exceed 18 hours.  2. Prior to intravenous injection, obtain a baseline blood sample using one of the 3 described methods below (in-dwelling catheter, retro-orbital (RO) bleed, peripheral bleed). 3. Administer a 0.25-1g/kg intravenous glucose bolus via the tail vein. Timed sampling will begin at the time of glucose administration. 4. Obtain a blood sample at 0 (baseline), 1, 5, 10, 20, 30, and 50 minute time points following tail vein glucose injection. Specific time points can be modified as per the investigators needs.
?. Blood Sampling Methods:1. Awake Indwelling Catheter Method: a). Anesthetize the mouse with the anesthetic approved by the given parent protocol (either inhalant or injectable).  b). Place mouse in dorsal recumbency. c). Once in a surgical plane of anesthesia, clip the hair covering the designated surgical site and perform sterile scrub. d). Create a 1-1.5cm incision in the jugular furrow region and gently dissect away the subcutaneous tissue to expose the vessel of interest (either Jugular or Carotid, as defined in the parent protocol). e). Using non-absorbable suture, occlude the cranial and caudal end of the vessel to occlude blood flow.  f). Create a small incision in the vessel to insert the catheter. g). Stabilize the catheter in the vessel with non-absorbable suture.  h). Test the patency of the catheter by injecting and flushing lock solution. i). Tunnel the distal end of the catheter though the subcutaneous tissue and using a stab incision, exteriorize the end between the scapulae on the dorsum. j). Secure the catheter at the exit point with suture.  k). Close the incision at the jugular furrow using tissue glue, absorbable suture or wound clips.  2. Retro-orbital (RO) Blood Sampling:a). Lightly anesthetize mouse with inhalant anesthesia just prior to blood collection unless prolonged anesthesia is required; if prolonged anesthesia is needed, injectable anesthesia can also be used. b). Apply ophthalmic anesthetic to the eye that will be used for sampling. c). Using a micro-hematocrit tube, penetrate the intraorbital capillary plexus to obtain approximately 100uL of blood.3. Peripheral Sampling: a). Gently restrain mouse. b). Using a sterile lancet puncture the peripheral blood vessel of interest (tail, saphenous or submandibular).   c). Blood can be measured directly from the lanced site with a handheld glucometer or a hematocrit tube can be used to collect the blood droplet for future analysis.
TITLE: Corallochytrium limacisporum_culture method
AUTHORS: Meritxell  Antó

[STEPS]
?. [Growing medium]
BD Difco Marine Broth 2216 • Marine Agar 2216 https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/212185.pdf
?. [Growth conditions]
Temperature 23ºC
?. [Cryopreservation and recovery (from liquid cultures)]
Cryopreservation  (10%DMSO in Growing medium)1. Pre-grow the culture in appropriate growing medium until almost saturated but not too old.2. Mix 1.35mL of the culture with 150 ul of DMSO  in the screw-top cryovials.3. Transfer the cryovials to the Freezing container, Nalgene®Mr. Frosty unit. Place it in the -80ºC freezer and leave it overnight.4. Remove cryovials from the Freezing container and store in appropriate boxes for -80ºC.Recovery1. Fill a flask with 10mL fresh Growing medium.2. Take out the frozen stock from the freezer, carry it to the culture hood in a cryo box to prevent it from thawing.3. With a pipette tip (or a loop), take a chunk of the frozen stock and resuspend it in the culture flask (~10uL is probably sufficient). Take the frozen stock back to the -80ºC as rapidly as possible.4. Let it recover for at least 3-4 days and proceed with another transfer in a fresh medium even if the cells are not completely grown it will help to dilute the remaining DMSO from the cryopreservation, the time will vary depending on the amount of the initial inoculum and the culture type.*NOTE: because of DMSO some precipitates may appear in the culture.All protists must be stored in liquid nitrogen for long-term storage. Although storage at -80°C is sometimes sufficient for a temporary alternative.
TITLE: Odor Retention
AUTHORS: sdwalto, Jeffrey Kordower, Bryan_Killinger

[DESCRIPTION]
Odor retention test optimized for mice. This test assesses short term olfactory memory. Mice with olfactory impairments or deficits should score lower. 

[GUIDELINES]
ODERANT PAIRS: limonene (diluted 1:10) and carvone (diluted 1:10); Amylacetate (1:100) and Anisol (1:100); and propyl acetate (1:10) and Pentanal (1:10).
 
Notes: 
- Paper swabs soaked in a given odorant were used inside the cartridges. 
- Investigation time defined as the duration of active sniffing with the nose placed less than 1 cm away from the cartridge. 
 

[STEPS]
1. Prepare and empty rodent cage with no bedding. 
2. Prepare the odorants by diluting in mineral oil. (See guidelines)
3. Acclimate mouse to setup (empty cage with odor cartridges). Place mouse in the cage and expose to 2 empty odor cartridges on either side of the cage for 5 mins. 
4. Expose mouse to the same unfamiliar odor in both cartridges for 5 mins. (Acquisition)
5. After 6, 16 or 30 mins post-acquisition, expose the mouse to a novel odorant and the familiar odorant from Step 4 and record the time spent investigating both the novel and familiar odorant for all trials. (Total investigation time capped at 1 min). (Recall)
6. The preferred odor was recorded and the preference was calculated based on the recall trial.
TITLE: cDNA protocol - Thermo Scientific RevertAid RT 
AUTHORS: Lauren  Ponisio, Jocelyn Zorn

[DESCRIPTION]
cDNA protocol - Thermo Scientific RevertAid RT

[STEPS]
SECTION: Prepare workspace
1. Thaw RNA and reagents on ice. Label four foil lids: one to replace RNA lid, one for reaction and two for aliquots
SECTION: Prepare workspace
2. UV sterilize 3 RNase free PCR plates, 8 boxes 10uL filter tips, and DEPC treated H2O
SECTION: Prepare workspace
3. Once thawed, flash spin down RNA in plate centrifuge and spin down reagents in mini centrifuge
SECTION: cDNA conversion
4. Add 1 µL RNA (.1 ng -5 µg) to empty PCR plate

SECTION: cDNA conversion
5. Add 1 µL   

 

SECTION: cDNA conversion
6. Add 10 µL DEPC treated H2O
 


SECTION: cDNA conversion
7. Add 4 µL 5X reaction buffer

SECTION: cDNA conversion
8. Add 1 µL   (20 U/uL)

SECTION: cDNA conversion
9. Add 2 µL   

SECTION: cDNA conversion
10. Add 1 µL   

 

SECTION: cDNA conversion
11. Mix gently with pipette and centrifuge briefly on "short"
SECTION: cDNA conversion
12. Place in thermocycler and run program "cDNA"
 

SECTION: cDNA conversion
13. Aliquot product into microplates and seal with labeled foil lid
SECTION: cDNA conversion
14. Store at -20 °C for up to 1 week or -80 °C for longer term storage
TITLE: pTpPuc3_TpSil3p-eGFP
AUTHORS: Jernej Turnsek

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Map and nucleotide sequence of the conjugation episome used to generate a TpSIl3p-EGFP expressing T. pseudonana strain reported in Faktorová D. et al. </div></div>

[STEPS]
?.
TITLE: Fecal Pellet Composition
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to measure the water content of mouse fecal pellets over 1 hour.

[STEPS]
1. Place mice in a clean cage and collect their fecal pellets produced over 1 hour.
2. Return mice to their home cage and weigh the fecal pellets (wet weight).
3. Desiccate the fecal pellets at 65oC for 19 hours then reweigh the fecal pellets (dry weight).
TITLE: Mitochondrial complex activity assays
AUTHORS: michela.deleidi, María José Pérez J.

[DESCRIPTION]
Mitochondria complex activity assays measure the activity levels of the different complexes of the mitochondrial electron transport chain (ETC).

[STEPS]
SECTION: Mitochondrial complex activity assays
1. Isolate mitochondria from HEK cells, iPSC-derived neurons, or midbrain organoids using the Qproteome Mitochondrial isolation kit  (QIAGEN, Cat. No. / ID: 37612) according to manufacturer's instructions. 
SECTION: Mitochondrial complex activity assays
2. Measure Complex I (NADH oxidase/coenzyme Q reductase) using the MitoCheck Complex I Activity Assay kit (Cayman Chemical, cat# 700930). 
SECTION: Mitochondrial complex activity assays
3. Determine the rate of NADH oxidation, which is proportional to CI activity, by a decrease in absorbance at 340 nm over 15 min in the presence of ubiquinone and potassium cyanide to inhibit complex IV and prevent oxidation of ubiquinone. 
 

SECTION: Mitochondrial complex activity assays
4. To this end, resuspend 3 mg of purified fresh mitochondria in 200 µL of MAS buffer (70 millimolar (mM) sucrose, 220 millimolar (mM) mannitol, 5 millimolar (mM) KH2PO4, 5 millimolar (mM) MgCl2, 1 millimolar (mM) EGTA, 2 millimolar (mM) HEPES pH 7.4)  and seed in XFpSeahorse microplates. 
SECTION: Mitochondrial complex activity assays
5. Centrifuge the plate at 2000 x g, 5 min, 4 °C. 
SECTION: Mitochondrial complex activity assays
8. Calculate Complex I-, II-, and IV-dependent respiration by subtracting OCR values from the substrates (Pyruvate + malate + ADP for CI, Succinate + rotenone for CII and TMPD + ascorbic acid for CIV) subtracted from the ones from the inhibitors (rotenone for CI, antimycin A + rotenone for CII and azide for CIV). 
SECTION: Mitochondrial complex activity assays
6. Measure the OCR before and after the serial addition of pyruvate + malate (5 millimolar (mM) each) + ADP 3,5 mM or 1 mM Succinate + 4 micromolar (µM) rotenone, 4 micromolar (µM) rotenone + 8 micromolar (µM) antimycin A, 0,5 mM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, Santa Cruz Biotechnology) + 1 millimolar (mM) ascorbic acid, and 50 millimolar (mM) azide. 
SECTION: Mitochondrial complex activity assays
7. Following each injection, record three measurements for a total period of 15 min .
SECTION: Mitochondrial complex activity assays
9. Normalize the experimental values to the protein content per well via a BCA assay.
TITLE: OSU TriState SenNet lung explant from IPF patients acceptance criteria
AUTHORS: Sean D. Stacey, Brenda F. Reader, Lorena Rosas, Victor Peters, Ana L. Mora, Mauricio Rojas

[DESCRIPTION]
This protocol describes the criteria required for inclusion of human lung specimen explants from idiopathic pulmonary fibrosis (IPF) diagnosed patients in Comprehensive Transplant Center Human Tissue Biorepository and TriState SenNet Biospecimen Core at The Ohio State University as part of the Cellular Senescence Network Program (SenNet). These criteria are based off of the CTC Human Tissue Biorepository SOP:009 Tissue Collection for Explant and Donor Samples. When processing explanted, diseased tissue, look for the most representative of that disease. Refer to the CT scan or Chest X-ray on the committee review to locate most diseased areas.

[STEPS]
SECTION: Inclusion Criteria
1. Transplant and surgical: 18-80 years of age, patient at OSUWMC, referred to OSUWMC for transplantation or has already undergone transplantation, able to understand and sign the TTCP Informed Consent and Research Authorization form, negative for HCV, HBV, and HIV, and has a pathology-confirmed diagnosis of IPF.Due to the nature of IPF pathogenesis, we remove specimen from both upper and lower lobes of a single lung. When processing explanted, diseased tissue, we choose samples from section of the lung that is the most representative of that disease. We refer to the CT scan or chest x-ray to locate these areas.
SECTION: Inclusion Criteria
2. In the case of IPF, we look for the following characteristics:
a)tissue is fibrotic, 
b)tissue shows thickening and scarring, 
c)tissue feels hard and stiff to the touch.
SECTION: Exclusion Criteria
3. 17 years of age and younger and unable to understand and sign the TTCP Informed Consent and Research Authorization form directly.
TITLE: Behavior Tracking with Running Wheels
AUTHORS: Victoria Vance, Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
This protocol describes tracking and analyzing behavior in mice using wireless running wheels. Because these running wheels are able to be placed inside an animal's home cage and continuously collect data, this method allows for the detection of activity throughout both the light and dark cycle.

[STEPS]
SECTION: Setting Up Wheels
1. Single-house mice in new cages containing activity wheels at least 3 days prior to beginning drug/vehicle to collect baseline data.
SECTION: Setting Up Wheels
2. Place clear wheel base holder on cage floor. If the experiment involves providing medicated water, place water bottle in the cage with sufficient autoclaved water. Note that excessive bedding crowds the cage and can cause water leaks.
SECTION: Setting Up Wheels
3. Turn on the wheel base and ensure that it connects to the Hub. Connect the base to the orange wheel and test that it properly detects wheel spins by manually spinning the wheel and monitoring the spin count. If the wheel connects and counts properly, place the wheel in the cage.
SECTION: Setting Up Wheels
4. Label each cage with the wheel number, put mouse in cage, and place on rack. If providing medicated water, ensure the mouse does not have access to other water sources, such as a LixIt spout.
SECTION: Setting Up Wheels
5. If cages cannot accommodate both wheels/water bottles and a food rack, provide food on the cage floor.
SECTION: Setting Up Wheels
6. Start data acquisition.
SECTION: Setting Up Wheels
7. Monitor Hub connection daily, replacing activity wheel batteries as needed. Change cages every  two weeks.
SECTION: Setting Up Wheels
8. At the end of each experiment, end data acquisition and clean all components with 10% bleach  and water.
SECTION: Wheel Analysis
9. Export wheel data file from Master Hub as an Excel sheet (Wheel Manager -> Export Data).
SECTION: Wheel Analysis
10. To analyze data in Excel, take the average data from the light and dark cycles in the vivarium (e.g.  7pm-7am for each night, and 7am-7pm for each day).