eamag/protocols-with-steps huggingface dataset:
TITLE: HuBMAP - Tissue Processing with Paraffin Infiltration
AUTHORS: Marda Jorgensen, Franchesca Farris, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the automated processing of formalin fixed HuBMAP specimens for paraffin embedding.</div></div>

[STEPS]
?. [Processing Selection & Initial Steps]
Place dividers in processor sample basket and load cassettes. No more than 10 cassettes can be added to each partition. Cover the basket with the basket lid.
Perform transfer of cassettes in the hood to avoid inhalation of the 70% ethanol that the cassettes are held in prior to procesing
?. [Processing Selection & Initial Steps]
Open the retort and place the basket inside. Rotate handles until they lay flat against the sides of the chamber. Close and latch retort
?. [Processing Selection & Initial Steps]
Press "Tissue Processing" on Main Menu. Enter desired program number (program used listed below). Select Immediate Start or Delayed Start to control the begining of processing so that completion coincides wih availability to embed at termination.
Appendix 1Paraffin Fixation and Processing Program   ABCDEFG1  Station #    Solution*    Conc.    Time    Set Temp.    P/Vacuum    Mixer  2  1    EtOH    70%    Hold**    _________    off    off  3  2    EtOH    70%    0:35    _________    P/V    slow  4  3    EtOH    80%    0:35    _________    P/V    slow  5  4    EtOH    95%    0:35    _________    P/V    slow  6  5    EtOH    95%    0:35    _________    P/V    slow  7  6    EtOH    100%    0:35    _________    P/V    slow  8  7    EtOH    100%    0:35    _________    P/V    slow  9  8    Xylene    100%    0:45    _________    P/V    slow  10  9    Xylene    100%    0:45    _________    P/V    slow  11  10    Paraffin    _________    0:45    60⁰C    P/V    slow  12  11    Paraffin    _________    0:45    60⁰C    P/V    slow  13  12    Paraffin    _________    0:45    60⁰C    P/V    slow  14  13    Paraffin    _________    0:45    60⁰C    P/V    slow  15                                   *Maintenance if performed according to manufacturer’s recommendation.**Duration of holding time dependent on desired end time to start embedding.
?. Ensure that the retort is drained and the Clean Cycle has been run.  If not, perform Clean Cycle.
?. [Processing Selection & Initial Steps]
When processing is completed remove basket and press "Drain Retort"
?. [Processing Selection & Initial Steps]
Transfer cassettes from the processing basket to the embedding station paraffin chamber.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Description file for PLOS PONE-D-17-43572 - EMID:f954815f1ce44492</p>
<p>All speech samples are in samples.zip directory, all are 48kSa/s, 16 bit mono, to be played at -26dBoV corresponds to diotic 73dB SPL. Available at</p>
<p>https://www.dropbox.com/s/xme4tu1u9rhwvmx/samples.zip?dl=0</p>
<p> </p>
<p>Comparison_LOAD_complete.xls file SHEET COMPARISONS contains the subjective data of both tests - test with no load : Test A, test with parallel task: Test B.<br />Each line corresponds to one condition. Conditions are defined by CODER, PLS (Packer Loss Suppression), Distractor (Type of Background Noise), SNR (Signal-to-Noise ratio in dB, Acctive speech level measured via ITU-T P.56) as indicated in columns W,X,Y and Z.</p>
<p>For each of 22 conditions and each test A and B, S-MOS, N-MOS and G-MOS data are reported including their CI95 (confidence interval at 95% probability level).</p>
<p>Comparison_LOAD_complete.xls file SHEET DATA_SUMMARY_LOAD and SHEET DATA_SUMMARY_NO_LOAD contain the detailed data results for all samples. Columns C,D,E,F,G and H describe per-sample analysis results, while I,J,K,L,M,N refer to per-condition analysis. Each condition is represented by four sentences, while each reference condition (lines 41...52) are represented by one sentence only.</p>
<p> </p>

[BEFORE_START]
<p>All subjects must be instructed as per P.835 and short trial session (cca 5 samples) must be assessed withour recording the results.</p>

[GUIDELINES]
<p>as per ITU-T P.835</p>

[STEPS]
TITLE: Guide Pratique sur l&#39;Échantillonage des Animaux Vivants (Domestiques, Peridomestiques et Sauvages) pour la Surveillance des Maladies Infectieuses
AUTHORS: Stefano Catalano

[DESCRIPTION]
Les cas de maladies causées par des agents pathogènes particulièrement dangereux ou mal diagnostiquées présentent des risques pour la santé publique et la prolifération. De nombreuses études ont démontré que dans les milieux à faibles ressources, et en particulier là où le paludisme est endémique, les patients qui se présentent dans les établissements de santé avec une maladie fébrile peuvent ne pas recevoir un diagnostic précis du fait, entre autres, de lacunes dans les protocoles différentiels et/ou d’une indisponibilité de tests diagnostiques. En Guinée, lorsque les patients se présentent au niveau communautaire ou préfectoral avec une maladie fébrile, les faiblesses des protocoles cliniques différentiels se traduisent généralement par le diagnostic d'une infection courante, notamment le paludisme et parfois la typhoïde, sans qu'aucun autre test ne soit effectué. À ce titre, la maladie fébrile aiguë représente une charge non quantifiée et indifférenciée pour le système de santé guinéen. Les agents pathogènes à l'origine d'épisodes fébriles dans la population, et les voies de transmission zoonotique qui peuvent les conduire, ne sont pas identifiés de manière systématique ni exhaustive. Cette situation constitut un défi spécifique pour les autorités de santé publique. Fever Project est un projet de recherche d'une durée de trois ans et avec le but de renforcer les capacités pour la réduction de la menace en Guinée et d'améliorer la sécurité sanitaire grâce à une approche intégrée de la santé humaine et animale en identifiant des étiologies à conséquences élevées des maladies fébriles aiguës chez l'homme. Les objectifs du projet sont les suivants: I) identifier les agents responsables de la maladie fébrile aiguë chez l'homme en Guinée; II) découvrir des preuves de la transmission d'agents pathogènes zoonotiques à conséquences élevées associés à une maladie fébrile aiguë; III) étudier les facteurs de risque liés aux maladies fébriles aiguë chez l'homme, y compris l'exposition aux animaux domestiques et péri-domestiques; et IV) améliorer les capacités de détection des agents pathogènes à conséquences élevées associés aux maladies fébriles aiguës.

[STEPS]
1.
TITLE: Passaging of Organoids in Matrigel
AUTHORS: Annika Fendler

[DESCRIPTION]
This protocol is for passaging prostate cancer organoids in matrigel. Protocol modified from Drost et al. (see citation below)

[STEPS]
SECTION: Before start
1. Prepare sufficient medium for passaging and prewarm immediately before use
SECTION: Before start
2. Thaw   on ice o/n

SECTION: Before start
3. Warm   to 37°C before use

SECTION: Before start
4. Coat 15 ml Falcons with 1% BSA o/n
5. Coat tips before use in 1% BSA by pipetting up and down a few times
SECTION: Dissociate organoids
7. Break up matrigel lense by pipetting up and down with a P1000 for approximately 10 times and transfer all content of a well to a coated 15 ml Falcon.
Repeat with all remaining wells
SECTION: Dissociate organoids
8. Once the content of all wells is transferred to the Falcon, pipette the whole suspension up and down a few more times to further break up the matrigel.
SECTION: Dissociate organoids
9. Add up to 10 mL  of PBS

SECTION: Dissociate organoids
10. 500 x g, 5 min, 21 Room temperature 
The higher speed helps to pellet the cells below the matrigel layer. Otherwise they get trapped in the matrigel and it is easy to suck them up with the supernatant
SECTION: Dissociate organoids
11. Mark the top of the matrigel pellet. Remove as much supernatant as possible without disturbing the matrigel.
SECTION: Dissociate organoids
12. Add 5 mL  PBS

SECTION: Dissociate organoids
13. 500 x g, 5 min 

SECTION: Dissociate organoids
14. Remove supernatant as described above
SECTION: Dissociate organoids
15. Add1-2 mL prewarmed   and pipette up and down to make sure that the cell pellet is well resuspended. 

SECTION: Dissociate organoids
16. Place falcon in incubator for 10 min to 15 min 

SECTION: Dissociate organoids
17. Pipette up and down for 10 times every 5 min and check the progress under the microscope
SECTION: Dissociate organoids
18. When only small clusters and single cells remain, add 5 mL  PBS

SECTION: Dissociate organoids
19. 400 x g, 5 min 

SECTION: Dissociate organoids
20. Remove supernatant and resuspend pellet in another 5 mL  PBS

SECTION: Dissociate organoids
21. 400 x g, 5 min 

SECTION: Dissociate organoids
22. Remove as much supernatant as possible and keep the Falcon tube on ice until ready.
SECTION: Seed cells in organoids
23. Depending in the passaging ratio, decide how many wells you want to seed the cells into and calculate the necessary amount of matrigel.



 

SECTION: Seed cells in organoids
24. Add matrigel to the cell pellet and carefully resuspend cells. Avoid formation of bubbles.
SECTION: Seed cells in organoids
25. Carefully add a matrigel drop to the center of the well.
SECTION: Seed cells in organoids
26. Flip the plate and let the matrigel solidify at 37 °C in the incubator for 15 min 

SECTION: Seed cells in organoids
27. Carefully add prewarmed medium on top of matrigel lense.
SECTION: Seed cells in organoids
28. Incubate at 37 °C in the incubator to expand cells.

6. Take pictures of the organoids to count organoids per well and measure the size of the organoids
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol presents a Gibson Assembly design for highly efficient construction of diatom episomes. We regularly observe &gt;90% efficiency (efficiency = % of screened bacterial colonies containing the desired construct) following the steps presented here.</p>

[BEFORE_START]
<p>This is not a Gibson Assembly tutorial such as <a href="http://www.openwetware.org/wiki/Janet_B._Matsen:Guide_to_Gibson_Assembly " target="_blank">this here</a>, but a brief overview of a Gibson Assembly design which significantly increases the likelihood of correct assemblies. We also assume familiarity with diatom episome structure as reported in the literature.</p>

[GUIDELINES]
<p>[a] This protocol has been tested with pPtPBR1 episome backbone in the following two contexts:</p>
<p> </p>
<p>1. streamlining construction of a variety of expression cassettes with NR (nitrate reductase) promoter and terminator sequences</p>
<p>2. transferring an existing expression casette from one episome to another</p>
<p> </p>
<p>pPtPBR1 - as well as other - diatom episomes can be obtained via <a href="https://www.addgene.org/Philip_Weyman/" target="_blank">addgene</a>. </p>
<p> </p>
<p>[b] We recommend using <a href="http://nebuilder.neb.com/" target="_blank">NEBuilder</a> to design Gibson Assembly primers.</p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #222222; font-family: arial, sans-serif; font-size: 12.8px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; text-decoration-style: initial; text-decoration-color: initial; display: inline !important; float: none;">This is a collection of protocols that accompany the Yuanyuan Fu, et. al 2017 <em>GigaScience</em> publication "Draft genome of the Tibetan medicinal herb, Rhodiola crenulata".</span></p>

[STEPS]
TITLE: Simulating colonic tissue mechanics using a structure-based material model in Abaqus
AUTHORS: Bhavesh Patel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The structure-based material constitutive model typically used for the colon tissue (DOI:10.1016/j.jmbbm.2017.08.031, DOI:10.1016/j.jmbbm.2013.02.016) is not readily available in the Finite Element software Abaqus, which is commonly used for biomechanical simuations. In this protocol, we provide step-by-step guidelines to use such material model in Abaqus using the User Subroutine implemented by the autor.</div></div>

[STEPS]
?. [Generate input file]
Once your model setup is completed (geometry definition, orientation assigment, meshing, etc.), go to the "Job" module and click on "Create Job".
?. [Specify material parameters in input file]
The structure-based material constitutive model typically proposed for the colon is shown below. The values of your material parameters C_10, k1_l, k2_l, k1_s, and k2_s (pre-determined from mechanical testing or gathered from pre-published work) must be specified in the input file. To do so, open the input file with any text editor (e.g. Notepad).
?. [Assign user material model]
Enter "UANISO_HGO" for the name of the material in the "Edit Material" dialog box that opens, and select General -->User Material.
?. [Assign user material model]
Click "OK" and assign the material to the desired sections of your model, as you would typically do with any material property.
?. [Assign user material model]
Go the the "Property" module of Abaqus and click on "Create Material".
?. [Generate input file]
Give a job name (e.g., MyJob) and select "Continue", then "OK" in the next dialog box.
?. [Generate input file]
Your job should be now listed in the "Job Manager" dialog box. Select it and click "Write Input". An input file (MyJob.inp) must have been created in your Work Directory.
?. [Specify material parameters in input file]
Go to the MATERIALS section of the file. You shoud see the material UANISO_HGO (defined in step 2) listed here.
?. [Specify material parameters in input file]
Specify the value of material parameters material parameters by entering the following lines below the name of the material (copy/past this and replace material parameter names by the desired values):*Anisotropic Hyperelastic, user, formulation=INVARIANT, type=INCOMPRESSIBLE, local direction=3, properties=8C_10, 0., k1_s., k2_s, 0., k1_l, k2_l, 0.
?. [Run simulation]
Go back to the "Job" module of Abaqus and create a new job.
?. [Run simulation]
Select "Input file" under "Source" and browse to the location of your input file then click "Continue".
?. [Specify material parameters in input file]
Save the input file and close it.
?. [Run simulation]
Go to the "General" tab of the new dialog box "Edit Job". Under "User subroutine file", browse to the file "uanisohyper_inv_colon.for". Complete any other specification you may want to include and click "OK".
?. [Run simulation]
You can now select your job from the "Job Manager" dialog box and click on "Submit" to run your simulation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Describes the library preparation for Nanopore sequencing from low input DNA 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: Yale - Blood and Urine Creatinine
AUTHORS: Gary Cline, John Stack

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure followed to detect the concentration of creatinine in serum, plasma, and urine.</div></div>

[STEPS]
?. Fill 1.5mL eppendorf tubes with 80µL of acetonitrile.
?. Add sample:      a. Use 20µL of plasma or serum/tube      b. Use 6 µL of urine/tube
?. Add 6.5 µL of d3-creatinine (5mg/dl) to each tube.
?. Vortex each sample for 5 seconds.
?. Spin each sample at 4˚C for 10 minutes
?. Transfer supernatant to LC/MS/MS vials.
?. Dry samples in speed vacuum.
?. Resuspend pellet in 75µL of LC/MS/MS Buffer
?. LC/MS/MS (liquid-chromatography/ tandemmass spectrometry) analysis: LC- Column: Isocratic using a Hamiltion PRP-X200 column. Mass Spectroscopy: MRM mode with parent/daughter ion pairs of 114/44 for creatinine and 117/47 for d3-creatine. (Ref: Takahashi N, Boysen G, Li F, Li Y, Swenberg JA. Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate. Kidney Int. 71: 266-271, 2006.)
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 the perfusion apparatus and place the tube into 1X Phosphate-Buffered Saline [PBS] 


3. Wash the system for 1 round, until the tube is saturated with 1X PBS 



4. Saturate anesthetic chamber with Isoflurane



5. Place the mouse in the anesthetic chamber for about 1 minute 



6. Remove the mouse from the anesthetic chamber and check reflexes by pinching the foot



7. If the reflex is active, place the mouse back into the chamber. If not, proceed with the perfusion



8. Grip the skin on the chest with forceps, and 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 a 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 the needle from the heart
TITLE: Transcriptome annotation
AUTHORS: Rafael Rodrigues Ferrari, Thiago Mafra Batista

[DESCRIPTION]
This protocol provides detailed, step-by-step instructions for students and researchers to annotate transcriptomes. In this tutorial, we will follow the Trinity -> TransDecoder -> Trinotate pipeline, using the SwissProt and Pfam databases for functional annotation of protein-coding transcripts.

[STEPS]
SECTION: FINDIG CODING REGIONS WITHIN TRANSCRIPTS
1. ****TransDecoder (https://github.com/TransDecoder/TransDecoder/wiki)****

***Extracting the long open reading frames (ORFs)***

**Prepare a .pbs file to run the analysis remotely on Sagarana**
 
***Including homology searches as ORF retention criteria***

**BlastP search**

*Prepare a .pbs file to run the analysis remotely on Sagarana*
 
**Pfam search**

*Download the Pfam database (Pfam-A.hmm)*

$wget ftp://ftp.ebi.ac.uk/pub/databases/Pfam/current_release/Pfam-A.hmm.gz

*Decompress the file

$gzip -d Pfam-A.hmm.gz

*Index the database*
 
*Prepare a .pbs file to run the analysis remotely on Sagarana*
 
***Predicting the likely coding regions***

#Run the 'TransDecoder.Predict' script in the same directory where the 'Trinity.fasta.transdecoder_dir' folder is located

**Without homology**
 
**With homology**

*BlastP*
 
*Pfam*
 
*BlastP + Pfam*
 

SECTION: FUNCTIONAL ANNOTATION
2. #Perform 'FINDIG CODING REGIONS WITHIN TRANSCRIPTS' first

****Trinotate (https://github.com/Trinotate/Trinotate.github.io/blob/master/index.asciidoc) (on kiko)****

***Generate databases***
 
****Blastn****

***Prepare a .pbs file to run the analysis remotely on Sagarana***
 
***Keep best hits only***
 
****Blastx****

***Prepare a .pbs file to run the analysis remotely on Sagarana***
 
***Keep best hits only*** 
 
****TMHMM (on kiko)****
 
****HMMER (on kiko)****
 
****SignalP (on kiko)****
 
****RNAmmer (on kiko)****
 
***Generating a .gene_trans_map***
 
****Populating the .sqlite file****

***Loading transcripts and coding regions***
 
***Loading BLAST homologies***
 
***Loading Pfam protein domains***
 
***Loading transmembrane domains***
 
***Loading signal peptide predictions***
 
***Loading rRNA gene predictions***
 
****Generate an output of Trinotate annotation report****
TITLE: Plasma Preparation 
AUTHORS: Sierra Simpson, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for preparing plasma for metabolomics studies / GWAS</div></div>

[STEPS]
?. Collect blood into the  EDTA coated microvette tube. Once spun, about half will be plasma, half will be whole blood. For initial harvest of blood,  we use retro-orbital blood sampling. This is done under general anesthesia ( isofluorane )     - The tip of the capillary tube is placed at the medial canthus of the eye under the nictitating membrane.    - A short thrust past the eye and the capillary will enter the sinus membrane which will have slight resistance.     - Apply slight pressure to the membrane and the blood will begin to flow into the capillary.     - Collect  200-400ul, then remove the capillary tube from under the eye and apply slight pressure to stop bleeding.     - Invert the tube 2-3 times to ensure the EDTA is evenly mixed in the sample, then move to step 2. For the final harvest of blood, we collect during the dissection of the animal. This allows us to directly collect blood from the circulatory system. The animal is first anesthetized with carbon dioxide, and then the heart is exposed.     - With an 18 gague needle, puncture slightly to the right side ( animals left)  of the apex of the heart.     - Apply slight backward pressure to the syringe to extract the blood.     - 2-3 mls is sufficient. The blood is transferred from the syringe to an EDTA coated tube.     - Invert the tube 2-3 times to ensure the EDTA is evenly mixed in the sample, then move to step 2.
0 Room temperature
?. Maintain the whole blood sample at room temperature, then aliquot the sample into a single tube or multiple tubes depending on the sample size. Freezing or extended time on ice will result in hemolysis.
?. Spin the whole blood samples for 10 min at 2000 g at RT to pellet the erythrocytes.
22 Room temperature
?. Immediately transfer 500 µL aliquots of the plasma in to fresh 2 mL labeled eppendorf tubes using a pipette with appropriate filter tip. Care should also be taken to avoid taking any red blood cells over into the plasma. It is best to err on collecting less than contaminating with red blood cells.
?. Visualize the quality of the plasma on the hemolysis scale below and mark the color of the plasma according to the scale 0-6 on the GWAS Blood collection sheet. It is optimal that the score is less than "1", take extra notes if there is excessive hemolysis, or clotting in the sample.
?. Special care should be made to ensure that the identification on each vial is legible. Make sure that the pen you use is        permanent ink and will not wash off upon thawing.Make sure the tube is labeled with an "A" for BSL, a "B" for final blood
?. Samples should be stored at -80 °C until analysis and all transportation should be carried out under dry ice.
TITLE: Phosphorus Extraction - Olsen Method
AUTHORS: maggie.bowman

[DESCRIPTION]
The method is based on the extraction of phosphate from the soil by 0.5 N sodium bicarbonate solution adjusted to pH 8.5.

[STEPS]
1. Print the required labels needed for Bray extraction for each site, depth, and replicate. 
2. Weigh out 3 gof soil into labeled 50 mL centrifuge tubes.

3. Add20 mL of Olsen Extract solution (in cabinet under hood in 1521) using the pipet and electronic pipettor. 

4. Cap the samples and shake to mix. Add samples to foam block on shaker. 
5. Shake samples at  . 

6. Centrifuge samples (using centrifuge on west side of 1521) for 4000 rpm, 8 min. 

7. Pour off liquid into a clean and labelled 50 mL centrifuge tube.

8. Freeze samples on their side to prevent tube breaking in the -20 °C freezer (Staging Freezer 2) bottom shelf if unable to filter immediately.

2.1. For example: 1000S_PRS1_P_OLSEN_TOP_1
1.1. 1000S_SITE_OLSEN_DEPTH_REPLICATE
1.2. 1000S_SITE_Pext_OLSEN_DEPTH_REPLICATE
7.1. For example: 1000S_PRS1_Pext_OLSEN_TOP_1
9. Follow sample filtering protocol below when ready to filter extracts.