eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.</span></p>
<p> </p>
<p><span style="font-weight: 400;">This protocol describes the steps for performing the immunohistochemistry method with frozen tissue sections.</span></p>

[GUIDELINES]
<p>Tissue preparation is a key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, the protocol given here is intended as a starting point from which the experimenter must optimize as needed. All conditions should be standardized in order to ensure reproducible results. Keep in mind that you must be careful not to allow tissues to dry out at any time.</p>

[STEPS]
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TITLE: Staining cells with IncuCyte Cytolight Rapid Dyes for flow cytometry or fluorescent microscopy
AUTHORS: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Eric Czech, Jeff Hammerbacher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for labeling cells with IncuCyte Cytolight Rapid dyes from Essen Bioscience. We optimized the dye concentration for Jurkat cells, pmel-1 T cells, and B16F10 melanoma cells. </div></div>

[STEPS]
?. Harvest cells and wash with 1X PBS
?. Centrifuge
Centrifuge: 350 34
?. Add IncuCyte Dye For pmel-1 T cells: we determined that 0.11 µM of green dye was optimal We made our working stock 11 µM  (so for every 1 ml of cell suspension, we add 10 ul of working stock of dye)For Jurkat cells, 1 µM of green dye was optimal For B16F10 cells, 0.11 µM of red dye was optimal
?. Incubate the cells at   for  (we wrapped the tube in foil and placed it in a water bath).
37 °C
?. Resuspend cells in PBS at 1 million cells per ml
?. Add a 6-fold volume of complete media (containing serum) to bind the excess dye.
?. Centrifuge
Centrifuge: 350 34
?. Resuspend cell in complete media at the desired concentration. Two different cell types can be labeled with different dyes and then co-cultured and visualizedStained T cells can be visualized migrating in collagen gel
?. Assay the cells via flowcytometry or fluorescence microscopy. Flow cytometry: FITC for green dye; APC for red dyeFluorescence microscopy: FITC filter for green dye; Cy5 filter for red dye
TITLE: proteomics (mitochondria)
AUTHORS: Livia Hecke Morais, Baiyi Quan, Tsui-fen Chou

[DESCRIPTION]
Here we describe proteomics experiment with isolated mitochondrial extracts at the Proteome exploration laboratory (PEL) at Caltech by Baiyi Quan, Jeff Jones and Tsui-Fen Chou in collaboratin with Livia Hecke Morais and Sarkis Mazmanian.

[STEPS]
SECTION: Sample Preparation
1. 
      Proteins extracted from mitochondria are reduced using a final concentration of 5 mM of TCEP (tris(2-carboxyethyl)phosphine) at room temperature for 10 min.
      The proteins are further alkylated using a final concentration of 20 mM of CAA (chloro-acetamide) at room temperature for 15 min.
      A 25 ul aliquot of the lysate is acidified using 2.5 ul of 12% phosphoric acid. Using a pH test paper to make sure solution pH< 2.
      Combine 165 ul of the S-trap buffer (90% MeOH with 100 mM triethylammonium bicarbonate) with the acidified lysate.
      Transfer the combined colloid into the S-trap micro (Protifi, NY).
      Centrifuge the S-trap micro at 4000 g for 1 min at room temperature.
      Using 150 ul of the S-trap buffer to wash the S-trap and centrifuge at 4000 g for 1 min. Repeat the step two more times.
      Add 20 ul of 100 mM triethylammonium bicarbonate containing 20 ug of Trypsin into S-trap micro. Allow the incubation stay overnight (14-16 hr) at 37 C.
      After overnight incubation, directly adding 40 ul of the 50 mM triethylammonium bicarbonate and centrifuge at 4000 g for 1 min.
  Add 40 ul of 2% formic acid in water to the S-trap, and centrifuge at 4000 g for 1 min.
  Add 40 ul of 50% acetonitrile in water to the S-trap, and centrifuge at 4000 g for 1 min.
  Speedvac the sample.
  The sample is reconstituted in 20 ul of 2% acetonitrile and 0.2% formic acid in water and 500 ug of the peptide is used for LC-MS/MS analysis.
SECTION: LC-MS/MS
2.       An aliquot of sample containing 500 ug of the
peptides is subjected to the LC-MS/MS analysis. The sample is separated on an Aurora
UHPLC Column (25 cm × 75 μm, 1.6 μm C18, AUR2-25075C18A, Ion Opticks) using an
Easy-nLC 1200 liquid chromatography system. The gradient settings follows Table 1.
 

Mobile Phase A: 0.2% formic acid, 2% acetonitrile, and 97.8%
water.
Mobile Phase B: 0.2% formic acid, 80% acetonitrile, and
19.8% water.
      The sample is analyzed on a Thermo Q-Exactive HF
mass spectrometer using a data-dependent acquisition method. Detailed
parameters of the scans are listed in Table 2.
 

SECTION: Proteomic Data analysis
3.       The raw data generated by mass spectrometer is analyzed using Proteome Discoverer 2.5. The data is searched using the mouse proteome achieved from UniprotKB on 10/26/2020 (swissprot + trembl). The processing and consensus workflow are set according to Figure 1 and 2. The parameters are listed below in Table 3. All the parameters that are not mentioned are left defaulted.

 


 

SECTION: Proteomic Data analysis
4.  
 

      The protein list exported from PD results are
further analyzed using R scripts based on the TidyProteomics package (jeffsocal.github.io/tidyproteomics/articles/overview.html).
TITLE: Enterococcus faecalis protoplast generation
AUTHORS: Elizabeth Fozo

[STEPS]
?. [Protocol]
Grow OG1RF in BHI + condition until OD600 0.3. This works for as little as 10mls of culture.
Growing in 2% glycine will reduce “gluing” of cells, but massively increase generation time.
?. [Protocol]
Spin cells down and resuspend in half volume isotonic solution.
?. [Protocol]
Add 2mg/ml lysozyme and incubate for 60 minutes. More time may be required.
?. [Protocol]
Spin cells down and wash with isotonic solution, resuspending again in isotonic solution in half the volume of the original culture.
When resuspending cells after lysozyme treatment, pipette mix and refrain from over-vortexing as this will kill protoplast cells.
?. [Protocol]
Check for wall removal using Gram stain. (Do not move on until protoplast confirmation)
TITLE: Lignin and Optional Sugars Analysis for Woodchips
AUTHORS: Feyereisen, Klasson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lignin and Optional Sugars Analysis for Woodchips</div><div class = "text-block">SOP 005-S-GF-19</div></div>

[STEPS]
?. Prepare washed filter papers (VWR part # 28297-984)
?. Rinse the filter papers on a vacuum apparatus by flushing 3x with a 20 mL aliquot of nano-pure water
?. Place the rinsed filter paper onto an aluminum tray, up to 20 filter papers may be places onto one tray
?. Put the aluminum tray with rinsed filter papers into the muffle furnace at 550o C for 30 minutes
?. Take out the filter papers after 30 minutes, using heat resistant gloves and the tongs
?. Allow the aluminum tray and filter papers to cool for 1 hour at room temperature
?. Rinse the filter papers again on a filtration apparatus with 3 aliquots of nano-pure water
?. Place the filter papers onto another aluminum tray, and into an oven at 105o C overnight
?. Weigh the mass of the washed filter paper, and the mass of the filter paper plus aluminum tray
?. Weigh 250 – 300 mg of dried woodchips into a clean 250 ml beaker, add a clean glass rod to the beaker for later steps
?. Add 3 mL of 72% H2SO4 to the 250 mL beaker with pipettor while stirring the sample well, wear safety glasses, lab coat, and nitrile gloves while handling H2SO4
?. Place the beaker, sample, and glass rod into the vacuum desiccator and put under vacuum for 15 minutes
?. Take the beakers out of the desiccator and cover with aluminum foil
?. Place the covered beaker into a water bath at 30o C
?. Stir the contents of the beaker frequently over the 60 minutes with the glass rod
?. After 60 minutes, remove the beaker from the water bath
?. Add 84 mL of nano-pure water to the beaker, stir, and remove the glass rod free of any particles
?. Cover samples with aluminum foil and autoclave for 60 minutes at 120o C
?. Allow the beakers to cool to room temperature
?. Samples can sit here overnight at room temperature
?. If doing sugars, add 5 mL of 500 mg cellobiose solution (Internal Standard Solution)
?. Weigh a washed filter paper and labeled aluminum tin to the nearest 0.1 mg
?. Place the filter paper onto a filtration apparatus with a CLEAN Erlenmeyer vacuum flask
?. Pour the beaker through the filter paper and wash materials with 5 mL of nano-pure water
?. Transfer the filtrate to a 100 mL volumetric flask and dilute to the mark with nano-pure water.
?. Invert the volumetric flask 3 times to mix, and store 15 mL of filtrate into a clean, labeled scintillation vial
?. Scintillation vials can be frozen after the filtrate is diluted and collected
?. Wash the materials on the filter paper with nano-pure DI until the filtrate pH is neutral
?. Place the filter paper into the labeled tin and into the oven at 105o C to dry for 24-48 hours
?. Weigh 2-3 g of bulk material into a weighed and labeled aluminum tin for moisture determination
?. Ash the glass fiber filter paper/lignin sample (filter paper only, not the aluminum tray) in the muffle furnace (follow ashing SOP)
TITLE: CAMbank: SST Field Processing v1
AUTHORS: Eliah G Overbey, krr, jak, chm2042

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

Instructions for preserving: Serum and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.

Allow the blood to clot in an upright position for at least 30 minutes. 
For optimal biochemical results, centrifuge and aliquot SSTs within 3 hours of venipuncture.
SECTION: Centrifuge Settings
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 10 minutes
speed: 1300xg
SECTION: Centrifuge Settings
3. Place the SSTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Aliquot Serum
4. Carefully remove SSTs from the centrifuge and inspect for separation of red blood vs serum layers. 

Transfer SSTs to a sanitized laminar flow hood. 
SECTION: Aliquot Serum
5. Using a P1000, carefully aliquot serum into 2D barcoded tubes at 500µL each. 

Return 2D tubes with serum to the rack and place in the -80C freezer.
SECTION: Aliquot Serum
6. Recap and freeze the SST tubes to preserve DNA in the red blood cell pellet
TITLE: Ligation
AUTHORS: Brian    Teague

[DESCRIPTION]
A DNA ligase is an enzyme that forms phosphodiester bonds -- it can "glue" together two pieces of DNA. The ligase we're using comes from the T4 bacteriophage virus.

Why do we need to do a ligation anyway? Remember, we're going to use a Cas9 protein to "cut" your target gene in a yeast cell. We can program the Cas9 to cut a particular place using a "guide RNA." However, does Saccharomyces cerevisiae -- brewers' yeast -- make Cas9 or a guide RNA on its own? No! So we need to give the yeast cell a set of instructions for making the Cas9 protein and the guide RNA.

We do so by encoding those instructions on a plasmid -- a small circular piece of DNA -- and then putting that plasmid into the yeast cell. Then, the yeast cell "follows" the instructions to make the Cas9 protein and guide RNA. The plasmid we're making has several functional DNA sequences on it -- a diagram is below.

  

The functional sequences on the plasmid are denoted by colored boxes -- you can ignore most of them for now. Note, however, the boxes labelled Cas9 and Guide RNA -- these are the instructions for making Cas9 and the guide RNA. Remember, though, that each group is knocking out a different gene -- so each group needs a different plasmid!

The part that needs to be customized for each group is the "target sequence" in the above diagram. Your instructor started with a plasmid similar to the one above, but where the target sequence is instead replaced by gene for a green fluorescent protein (GFP) -- cells that express it turn bright green! They have cut out the GFP, and now you need to replace it with a targeting sequence for your gene, as in the diagram below.

   

One last thing. Where is your targeting sequence coming from? It's the annealed oligonucleotides from last time. You'll mix the plasmid backbone, your annealed oligos, the ligase enzyme, and a ligase buffer (which contains all of the chemicals the ligase needs to operate), and then the ligation will incubate at room temperature while the enzyme does its thing.

[BEFORE_START]
This protocol depends on the successful completion of the Annealing Oligonucleotides protocol, below:
 


[GUIDELINES]
This protocol involves pipetting very small quantities of liquid. Pipette carefully!

[STEPS]
SECTION: Ligation
1. Dilute the annealed oligonucleotides in   to make 100 µL of working stock at a final concentration of  

SECTION: Ligation
2. In the PCR tube, mix in order:
4 µL   
2 µL  of the L2-01 DNA plasmid backbone
 2 µL of the diluted annealed oligos
1 µL   
1 µL  

SECTION: Ligation
3. Flick the tube several times to mix the components, then spin briefly in the microcentrifuge to collect everything at the bottom of the tube.
SECTION: Ligation
4. Incubate at room temperature at least 30 min, and up to 120 min (longer is better).

SECTION: Ligation
5. Store at -20 °C
TITLE: General preparation of liposomes using probe-tip sonication
AUTHORS: Monica Rieth, Andrew Lozano, Jordan Grant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method outlines a general approach for preparing liposomes using probe-tip sonication.  The method has been optimized for the preparation of pure DPPC liposomes on a 25-mg scale and may require modifications as the quantity of lipid is altered or upon addition of other lipids and small molecules. </div></div>

[STEPS]
?. Lipids are typically stored at   and should be allowed to reach room temperature prior to working with them.For DPPC powder, carefully weigh out 25 mg on a clean analytical balance using clean, ungloved hands (to minimize static)
-20 °C
?. Carefully transfer the lipid to a clean 2.0 mL glass vial.  Add 2.0 mL of (0.2 um-filtered) buffer.  (20 mM HEPES, 100 mM NaCl, pH 7.4)
?. Samples were vortexed to mix and hydrate the lipid powder or dried film (if prepared from a chloroform solution lipids should be dried to a film under a stream of nitrogen gas).
?. To suspend the lipids more homogeneously and remove large particulates the mixture can be sonicated using a probe-tip sonicator (Fisher Scientific, Hampton, NH) set to 20% duty cycle with a pulse time of 2 seconds followed by a rest period of 2 seconds for a total sonication time of 2 minutes.
?. To prepare liposomes from this mixture, the cycle in step 4 should be repeated 3 additional times for a total of 4 cycles at 2 minutes total sonication time per cycle.  Total liposome preparation time is 8 minutes.
The rest period is important to avoid excessive heating of the sample.  Temperature should always be monitored closely.
?. Samples were centrifuged using a standard benchtop microcentrifuge at  for 3 minutes to remove residual titanium particles from the sonicator probe tip and un-reconstituted lipids.
?. Carefully remove the supernatant and transfer to a clean 2.0 mL Eppendorf tube.
?. Samples can be stored at   for up to 24 hours.  An additional centrifugation step should be carried out on samples that have been stored to remove any precipitated lipid.
4 °C
TITLE: SOP Template
AUTHORS: Catherine.Cook

[DESCRIPTION]
Study Title: Rapid Research in Diagnostics Development for TB Network (R2D2 TB Network) Study
SOP Title: Template
SOP ID: 
Division: Data, Lab, 
Effective Date:
Written by: 

[STEPS]
SECTION: Purpose
1. This document describes in detail the procedure for locating and measuring MUAC (Middle Upper Arm Circumference) during the baseline questionnaire, Follow-up visit 1 , and Follow-up visit 2  for R2D2 study participants
SECTION: Definitions
4. REDCap: Research Electronic Data Capture, a secure, online database that will be used for data entry and collection for the R2D2 TB Network study

 

SECTION: Background / Rationale
2. The Rapid Research in Diagnostics Development for TB Network (R2D2 TB Network) Study tests all participants for XXX s during the baseline visit
SECTION: Supplies and Materials
5. Tablet
 

2. 
SECTION: Data collection procedures
6. Intstructions 1
SECTION: Record the data
8. Record in Redcap form 
2. 
2. 
SECTION: Training implications
3. Study Coordinators
Research Assistants


SECTION: Data collection procedures
7. 
SECTION: Record the data
9. 
 

SECTION: Record the data
9.1. Critical step
TITLE: CSF Processing
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for processing CSF.

[GUIDELINES]
FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: CSF Processing
1. On dry ice, scan and position aliquots into the Freezerworks Inventory Program.
SECTION: CSF Processing
2. Stores aliquots in -80 °C freezer with 2 hours of LP draw.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a method for detecting Human Polyomaviruses (HPyVs) in our virome and whole metagenome datasets. For genome reference, download the HPyV genomes (in fasta format) from NCBI (nucleotide) using the search terms "Human Polyomavirus" AND "Complete Genome". This is used in the rest of the analyses. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplemental information available at:<br /><br />
<div class="page-content view-content">
<div>
<div class="view-block">
<div class="view-scroll">
<div class="vb-main">
<div class="b-start-block editable-content"><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a></div>
</div>
</div>
</div>
</div>
</div>

[GUIDELINES]
<strong>Required Software:</strong><br />
<ul>
<li>NCBI's BLAST+ v2.2.0</li>
</ul>
<br /><strong>Relevant Files</strong><br />
<ul>
<li>Output: HPyV_Detection </li>
</ul>

[STEPS]
?. 
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TITLE: Inducing proteostasis stress
AUTHORS: Louise Uoselis

[DESCRIPTION]
Inducing proteostasis stress in HeLa cells with G-TPP to analyse proteostasis protection and repair.

[STEPS]
SECTION: Day 1
1. Seed cells in standard growth media, aiming for a density of ~80-90% the following day. 
SECTION: Day 3
4. Harvest the G-TPP treated samples and DMSO control samples after the desired incubation period (eg. 2 – 12 h).
SECTION: Day 2
2. Feed cells for 1 h prior to starting treatment by aspirating the media from each well and replacing with a standard volume of growth media (eg. Feed with 2 mL of media if using a 6 well plate).
SECTION: Day 4
7. Harvest the 24 h R time point, at 24 h after the washout step (steps 5 – 6) was performed.
SECTION: Day 2
3. Aspirate the media from each well/plate, and replace with standard growth media that has had G-TPP added at a final concentration of 9 uM (for the DMSO control, add the same volume of DMSO as you added G-TPP to each well/plate).  
SECTION: Day 3
5. For samples to be analysed followed after G-TPP removal, aspirate the treatment media from each sample, wash each sample 3x in an ample amount of room temperature PBS (eg. 3 mL per well for a 6 well plate).
SECTION: Day 3
6. After 3X PBS washes, add an appropriate volume of standard growth media to each well/plate (eg. 2 mL of standard growth media per well for a 6 well plate). 
SECTION: Day 4
8. Replace the media on the plate for the 48 h R samples with an appropriate volume of standard growth media (eg. 2 mL of standard growth media per well for a 6 well plate).   
SECTION: Day 5
9. Harvest the 48 h R samples, 24 h after the samples had fresh standard growth media added (step 8).
TITLE: TS Spurrs - unstained FFPE on Thermanox coverslips (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
Processing unstained FFPE sections on Thermanox coverslips to Spurrs resin block for EM.

[GUIDELINES]
All step times are the minimum time required. Longer steps are acceptable.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:


SECTION: Dewax FFPE sections
2.   or X3B 5 min 

SECTION: Dewax FFPE sections
3.   or X3B 5 min 

4.   or X3B 5 min 

SECTION: Rehydrate
5. 100 % volume   5 min 

6. 95 % volume    5 min 

7. 70 % volume   5 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
8. 2.5% % volume     in 0.1 Molarity (M) Sorenson's phosphate buffer pH 07.2 300 mosmol/kg20 min.

SECTION: Conventional - with reduced times for 5um thick FFPE section
9. Wash buffer 5 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
10. 
1 Mass / % volume   in buffer30 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
11. 70 % volume    10 min 

12. 95 % volume   5 min 

13. 100 % volume   5 min 

14. 100 % volume    5 min 

15. 100 % volume Spurrs:100 % volume    in 1:1 ratio30 min 

16. 100 % volume Spurrs30 min 

17. 100 % volume Spurrs30 min 

18. Oven polymerise 65 °C
TITLE: Glycolysis stress test in organoids
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Glycolysis stress test in organoids 

[STEPS]
1.   were coated with Laminin solution in L15+Bicarbonate: final concentration of 10 μg/mL .

2. Day 35 midbrain organoids were placed in the plates and media was changed every other day.
3. On the day of the assay   supplemented with B27 + N2  was warmed at 37 °C  and the pH as adjusted to 7.4. And 175 ul was added to the organoid plates.

4. The plate containing the organoids were warmed at 37 °C  in a non-CO2 incubator for 30 min

5. Using the   kit the cartridges were loaded with loaded with Glucose (10mM), Oligomicyn (1uM) and 2-DG (50mM).

6. The run was done in 
 
 
7. After the run the organoids were lysed in RIPA buffer and total protein was quantified using BCA kit. 
8. The total protein was used to normalize the ECAR and OCR.