eamag/protocols-with-steps huggingface dataset:
TITLE: Tissue Processing
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Protocol for processing mouse tissue for immunohistochemistry.

[STEPS]
1. After completion of live experiments, anesthetize the mouse with institution-approved substance at correct dosage. 
2. Ensure the mouse is deeply anesthetized via the toe-pinch test and that there is no reaction. Place the mouse with its chest facing up above a basin to capture blood, and secure the paws so that the body doesn't move. 
3. While there is still a heart beat, open chest cavity using small scissors and forceps, making sure not to puncture organs.
5. Perfuse the mouse with 1x PBS to clear the blood.
6. Once blood has been cleared, perfuse the mouse with 4% PFA.
4. With a small needle/syringe attached to a pump, pierce the left (on your right side) ventricle. Pierce the left atrium to allow blood to flow through.
7. Once the whole body is perfused, dissect all needed tissues and organs.
8. Post-fix the dissected tissues in 4% PFA for 4-6 hours.
9. After post-fixation, gently dry the tissue. 
10. Embed each tissue to OCT block.
11. Section tissue at 40um thickness using cryostat.
TITLE: SA-B-Gal Activity
AUTHORS: Karyna Tarasova, Sinan Gültekin, iris.gerner Gerner, Florien Jenner

[DESCRIPTION]
To measure cellular senescence

[STEPS]
SECTION: Senescence associated SA--Galactosidase Activity
1. 
SECTION: Senescence associated SA--Galactosidase Activity
1.1. The cells were washed twice with ice cold PBS and then incubated with 60µL of cell lysis buffer
provided with the cellular senescence assay kit (Fluorometric format) (Cell Biolabs, USA) for 10 minutes at 4 ºC. 
SECTION: Senescence associated SA--Galactosidase Activity
1.2. The whole cell lysates were centrifuged 15 minutes at 14000 g at 4 ºC. Total protein concentrations were determined by Qubit Protein Assay Kit (ThermoFisher, Q33211) using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Singapore, shipped from Germany) according to the manufacturer’s instructions.
SECTION: Senescence associated SA--Galactosidase Activity
1.3. Following protein quantification, the assay buffer (Cell Biolabs, Inc., CBA-231, San Diego, USA) was added 1:1 to the cell lysates and incubated for 3h in the dark at 37 ºC.  
SECTION: Senescence associated SA--Galactosidase Activity
1.4. The reaction was stopped by adding 120 µL of the Stop solution (provided with the cellular senescence assay kit) per 30 µL of the reaction mixture. 
SECTION: Senescence associated SA--Galactosidase Activity
1.5. Fluorescence was measured using a plate reader at 360 nm (Excitation) / 465 nm (Emission). SA-b-Gal activity was normalized to total protein concentrations.
TITLE: Mitochondrial complex activity assays
AUTHORS: michela.deleidi, María José Pérez J., Hariam Raji, Pascale Baden, Federico   Bertoli

[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: Tetiaroa Vegetation Sampling
AUTHORS: Sage Moloney, Kenechi Elvis Obiorah, Erica Garibay, Allea Eimers

[DESCRIPTION]
Measurement of seedling recruitment for four native woody species, the shade cover of four herbaceous plant species, and coconuts on the Motu Ahuroa in Tetiaroa were conducted by students with the UC Berkeley Island Sustainability Program. The four woody species we observed were Pisonia, Pandanus, Guettarda, and Ipomoea. For the herbaceous species, we observed Asplenium, Boerhavia, Microsorum, and Portulaca. Once we completed our data collection in each transect, we compiled our data to see the effect of rat eradication programs on plant growth.





[BEFORE_START]
Thoroughly clean shoes and clothes to avoid transferring seeds to Ahuroa motu.
Arrive at pre-existing transects.
Understand what plant species are being studied and how to identify them.

[GUIDELINES]
Have picture identification of the woody and herbaceous plant species (especially the seedling stages as these may look different from adult plants)
Wear closed-toe shoes, long sleeves, and pants
Brush shoes off before arrival on the motu to prevent the introduction of invasive species
^ Do NOT bring fruits 
Stay hydrated, put on bug spray
Be cautious about where you step so you don't kill seedlings



[STEPS]
1. Find the marked transects using a GPS and/or flag markers.
2. Use the measuring tape to lay out a 25 m transect from the first marker to its respective end marker. 
3. Beginning with left or right side of the transect, measure a 1mx1m plot.
4. Within this 1m^2 plot:
4.1. For woody seedlings: identify and count seedlings in the given area, recording how many are present.
4.2. For herbaceous species: Estimate—using a visual assessment—plant coverage over the given area as a percentage.
5. Continue to next 1mx1m quadrant along the transect until you reach the end; total quadrants should equal 24.
6. Repeat steps 3-5 for the opposite side of the transect.
4.3. For coconuts: Record number of fallen coconuts and whether a fallen coconut is dead or sprouting. If sprouting, record the amount of leaves sprouting from the coconut.
TITLE: ONT Basecalling, Demultiplexing, and Analysis for Fungal Barcodes
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This protocol assumes that your MinION run has been completed and the data from the run has been saved. It should take you from raw data to useable FASTA files for each of your fungal barcodes.

[STEPS]
4. 
 

For a Flongle cell with 1.15Gb of bases and 1.18M reads, this command takes about 37 minutes to run. Example output:


 

Sometimes after the run I need to restart the CPU before this command runs successfully.
1. This protocol assumes the experiment name is "FirstRun."

Create a new working folder on the desktop. Ex - FirstRun. Within that create a new folder called "fast5," another called "Programs," and a final one called "NGSpeciesID."

I will start by copying all of the fast5 files from:

/var/lib/minknow/data/./FirstRun/CellName/long_unique_name/fast5 to the newly created fast5 folder on the desktop
5. 
 

6. 
 

 

7. 
 

3. Copy these Python scripts into the Programs folder you just created.

  
  
  

10. 
 
This should take about 3-4 minutes to run.

 

8. 
 

2. Create an index file from your extraction template papers. This will allow you to link all of your reads with the individual specimens. A template for 7 plates (672 specimens) can be found here:

  

This .xlsx is formatted to utilize the Lab Code and iNaturalist # columns as the only inputs. It will combine these and all of the other columns into a single cell - concatenating them all into the final file name. For the Lab Code, I will typically put these into the iNaturalist "Voucher Number(s)" Observational Field, and then export them all at once into a .csv from iNat. This allows me to simply copy and paste many iNat numbers at once, without ever needing to input any of the numbers manually.

After editing, save as a tab-delimited text file in the NGSpeciesID folder. You will need to remove most of the final columns from the template. The final output should be saved like this:

  

11. 
 
This program will take about 3-6 hours to complete.
12. 
 


9. 
 

 

Review the images that are generated. Ensure the quality scores of your run are in an appropriate range. For a 9.4.1 Flongle with Q20+ (V12) K12 chemistry, I typically get a peak in the 12-13 range.

  
Example of all outputs from this command:
TITLE: Protocol for Detection and Analysis of Cyclic RNA Using RT-PCR
AUTHORS: creative-biogene

[DESCRIPTION]
This protocol describes the design of divergent primers which face away from each other on the linear RNA, so that they can only amplify the circRNAs, and not the linear RNAs with the same sequence.

[STEPS]
SECTION: Experiment Summary
1. This protocol describes the design of divergent primers which face away from each other on the linear RNA, so that they can only amplify the circRNAs, and not the linear RNAs with the same sequence. The PCR amplicon for the detection of circRNAs using divergent primers spans the backsplice junction of circRNAs. This method has been successfully used in several studies for the detection and quantification of circRNAs.
SECTION: Equipment
2. Manual pipettes (set of 2 µl, 20 µl, 200 µl and 1,000 µl), cell scraper, vortex mixer, UV transilluminator, microvolume UV-Vis spectrophotometer, PCR strip tube rotor, mini centrifuge C1201, 96-well thermal cycler, OwlTM EasyCastTM B1 mini gel electrophoresis systems, gel imaging system, MPS 1000 mini plate spinner, QuantStudio 5 eal-Time PCR system.
SECTION: Procedure
3. A. Divergent primer design
1. Get the mature sequence of circular RNA (https://genome.ucsc.edu/) using the genomic coordinates.
2. As shown in Figure 1, make the PCR amplicon template by joining the 100 nt sequence from the 3' end to 100 nt sequence at the 5' end of the circRNA.
TITLE: Prepare bovine coronavirus (BCoV) solution
AUTHORS: Adélaïde Roguet, Shuchen Feng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Prepare bovine coronavirus (BCoV) solution from vaccine</span></div></div>

[STEPS]
?. [Prepare the BCoV solution]
Resuspend one vial of using 1 to 3 mL of 1xTE buffer.
?. [Prepare the BCoV solution]
Gently vortex, avoiding foaming.
?. [Prepare the BCoV solution]
Aliquot 40-200 µL of the BCoV solution into 600 μL low-binding tubes.
The volume aliquoted depends on the number of samples that have to be processed at once, to avoid freeze/thaw aliquots more than twice.
?. [Prepare the BCoV solution]
Store the aliquots at -80ºC.
?. [Nucleic acid extraction]
Thaw on ice a BCoV aliquot
?. [Nucleic acid extraction]
In duplicate, transfer 5 µL of the BCoV solution into a 2-mL tube containing 500 µL of warmed PM1 buffer solution  (provided in  ) + 5 µL of beta-mercaptoethanol..
?. [Nucleic acid extraction]
Extract nucleic acids of the duplicates
{"blocks":[{"key":"bca88","text":"Total nucleic extraction from wastewater using RNeasy PowerMicrobiome kit (qiagen)","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Titration]
Prepare a serial dilution for each duplicate to obtain the following dilution ratio: 1:1, 1:2, 1:8, 1:32, 1:128, 1:512, 1:2048Dilutions are performed using nuclease-free water in low-binding tubes.
?. [Titration]
Perform absolute quantification of PCR targets with the Droplet Digital PCR.
{"blocks":[{"key":"asjms","text":"Quantification by\u00a0Droplet Digital PCR (RT-ddPCR)","type":"unstyled","depth":0,"inlineStyleRanges":[{"offset":0,"length":48,"style":"bold"}],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Titration]
Formula to back-calculate the BCoV concentration in the initial solution:CopyPerMicroliterReaction * Reaction Volume / VolumeSample * DilutionFactor*ElutionVolume
?. The BCoV solution is ready to use.
The titer should be close to ~100,000 copies per microliter. If near 200,000 copies per microliter, the solution will have to be diluted before use.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>S1 Table. Pooled counts of Blue-fronted Amazons. Counts were performed in five roosts by month in the southern Pantanal of Brazil from July 2004 to July 2009. “Roost” is the roost id (1-5), “all.parrots” refers to the number of parrots arriving at roosts irrespective of the group size, “singles” refer to the numbers of singletons, “pairs” refer to numbers of dyads arriving the roosts. “Fledglings” are the number of young fledged in family flocks, “family.flocks” refers to the number of parrots arriving the roosts in small flocks of three to six birds, and “large.flocks” refer to number of the number of parrots arriving the roosts in large flocks (i.e. &gt; 7 parrots). (XLSX)</p>

[STEPS]
TITLE: Transcriptome profiling of brain and lung under Dip2a regulation
AUTHORS: rajiv kumar sah, Yang Anlan, Fatoumata Binta Bah, Salah Adlat, Ameer Ali

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Total RNA from brain and lung of E19.5 Dip2a</span><span style = "vertical-align:super;">-/-</span><span> and wildtype embryos was isolated and used as a input material for cDNA synthesis. The library preparation were send for sequenced and paired-ends were generated. Raw data of fastq format were processed and clean reads were obtained by removing adapter, reads containing poly-N and low quality reads. The clean reads were then mapped to  ouse reference genome. Gene annotation were done beased upon Nt, Nr, KOG/COG , EggNOG, KO and GO database. Quantification of transcription expression level was presented by FPKM. In order to identify diferentially expressed genes between WT and Dip2a</span><span style = "vertical-align:super;">-/-</span><span> embryos in brain and lung , DESeq from R package was used.  DEG enrichement was done based upon KEGG , GO and TF database.</span></div></div>

[STEPS]
?. [Sample collection protocol]
Dip2a heterozygous mice (Dip2a+/-) were intercrossed, female mice checked for presence of copulation plug (Vaginal plug) and designated as E0.5 day. At age of E19.5, pregnant dams were euthanized and embryos were collected on ice-cold 1X PBS. Brains and lungs were dissected out from each embryo and frozen immediately in liquid nitrogen and stored at -80oC.
?. [Nucleic acid extraction protocol]
Total RNA was isolated using RNAiso plus reagent (Takara, Dalian) according to manufacturer’s instruction. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
?. [Nucleic acid library construction protocol]
Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations.
?. [Normalization data transformation protocol]
Raw read (clean reads) were aligned under Tophat2 software and assembled with Cufflink software. Gene quantification was done based upon FPKM for each sample.
?. [Nucleic acid sequencing protocol]
The clustering of samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer’s instructions. Library were sequenced on an Illumina HiseqTM 2500 platform.
TITLE: Crystallisation of MERS-CoV Mpro
AUTHORS: blake.h.balcomb, Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson

[DESCRIPTION]
 The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus. 

[STEPS]
SECTION: Crystallisation experiment
4. Protein and buffer requirements:
32 µL17 mg/mL  
3.36 mL 
14.4 µL   seeds, dilution 1:1000

SECTION: Crystallisation experiment
6. Dispense 35 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL17 mg/mL    to each lens using the SPT mosquito.
Dispense  20 nL to each lens using the SPT mosquito.
Dispense  130 nL to each lens using the SPT mosquito.

Drop ratio: 15:13:2 ratio (150 nl   : 150 nl reservoir solution: 20 nl seeds)
Final drop volume: 300 nl
SECTION: Crystallisation experiment
5. Crystallisation screen composition:
0.2 Molarity (M)  Sodium malonate dibasic monohydrate
20% w/v PEG 3350

Stock solutions used:
JCSG+ condition 2-30

 

SECTION: Crystallisation experiment
8. Crystal form after ~12 h.
 


SECTION: Crystallisation experiment
7. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
2. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallisation experiment
3. Prepare seed stock:
 
1: 1000 dilution    seeds
SECTION: Data collection at Synchrotron
9. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
SECTION: MERS-CoV Mpro expression and purification
1. The protein used for crystallisation was expressed and purified using the following protocol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is used to clarify the process of RNA extration for our R. crenulata genome.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: General freezing protocol for HEK-Blue cells
AUTHORS: Andreas Sagen

[DESCRIPTION]
HEK-Blue is a product from Invivogen, which provide reporter cells for endotoxin-testing among others. Here is a generalized protocol for freezing cells with reporter characteristics. This protocol differ significantly from Invivogens recommendations and should be used with care, as it has not been tested on all the different cell lines provided by Invivogen.

[BEFORE_START]
Prepare freezing medium. Take care to heat and cool the necessary reagents.

[GUIDELINES]
The protocol has been proven to work with different HEK-Blue cells, but not all. Please follow the recommendations of Invivogen when buying a new batch, and test protocol when a sizable batch of HEK-blue cells are available in storage.

[STEPS]
SECTION: Concentrated complete freeze medium (CCMF)
1. Pre-prepare freezing medium. While you could create freeze medium outright, and suspend the cells directly in it, it is recommended to minimize the exposure time of cells with DMSO. To do this, the cells are initially resuspended and counted in serum-free medium. This give you time to create labels, etc. between resuspension and freezing.
SECTION: Concentrated complete freeze medium (CCMF)
1.1. In 70 mL DMEM, mix 20 mL FBS and 10 mL DMSO

 

Materials:
 
 
 
SECTION: Concentrated complete freeze medium (CCMF)
1.2. Aliquot complete growth medium in 10 mL units and store frozen at -20 °C

 

SECTION: Freezing procedure
2. When cells reach between 80-90% confluence, the freezing procedure can start

 

 

SECTION: Freezing procedure
2.1. Remove supernatant from monolayer. Store 2 mL or more for mycoplasma testing. Wash cell layer gently with PBS. Add 10 mL PBS to a T-75 equivalent flask, and incubate for 5 min. Lightly tap flask, and ensure cell detachment with microscope

Materials:
 
SECTION: Freezing procedure
2.2. Transfer suspension to a canonical centrifugation tube and centrifuge with 200 rcf, 2 min
SECTION: Freezing procedure
2.3. Decant supernatant and resuspend pellet in 1 mL warm serum-free DMEM

Materials:
 
SECTION: Freezing procedure
2.4. Create a 100 µL 10x dilution of suspension in PBS, and determine cell concentration.
Dilute cell suspension in warm serum-free DMEM to 2×106 live cells mL-1. Find volume DMEM to add with  , where   is the cell concentration found earlier
SECTION: Freezing procedure
2.5. Dispense 500 µL cell suspension to cryotubes or screw cap tubes. Create labels and attach to tubes. Lastly, add 500 µL ice-cold CCFM

 

Mix suspension, and transfer tubes to a cold block (Corning #432050) on ice. Transfer tubes or cold block to an insulated box (Corning #432021). Freeze at -80 °C for 1440 min. Transfer tubes to box, and store long-term in a vapor-phase nitrogen freezer or medium-term in a ultra-low temperature freezer
TITLE: cDNA Synthesis
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the synthesis of first-strand cDNA from total RNA using the Invitrogen SuperScript III Reverse Transcriptase system.</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]