eamag/protocols-with-steps huggingface dataset:
TITLE: Harvest of adherend cells from Cytodex® 1 beads
AUTHORS: David König, Kim Kremser, Sina Bartfeld, shirin.kadler

[DESCRIPTION]
Expanding adherend cells to quanteties suitable for cell therapy is a subject of increasing relevance.
Variouse cell types either autolog or allogen - could in future be used for medical purposes / in clinical application as a key component in therapy. This protocol discribes how to harvest adherend cells from dextran based Cytodex® 1 beads after expansion. Since other enzymatic schemes like Trypsin-EDTA or TrypLETM have shown low efficiency and yield - here we present an approach that is easy in implementation and delivers cells with high harvesting yield and high viability.

[GUIDELINES]
This protocol can surely be further improved but besides that is pretty much strait forward - crutial steps are the centrifugation and supernatent discartion where many cells can be lost if samples are not handled with due care.

[STEPS]
SECTION: sampling
1. withdraw homogenized cell solution from culture vessel
and transfer to centrifuge tube 10-50 mL 


SECTION: Micro carrier solving
2. add 0.1 % volume of dextranase (v/v) 




SECTION: Micro carrier solving
3. transfer samples to a tube rotator and place in an incubator  10 rpm, 60 min, 37 °C 

SECTION: Micro carrier solving
4. take out tubes and centrifuge 600 x g, 8 min 

SECTION: Micro carrier solving
5. carefully discard supernatant
SECTION: single cells
6. add a small volume 1-5 mL of 2x TrypLETM and completely solve the pallet

SECTION: single cells
7. take out tubes and centrifuge 600 x g, 8 min 
SECTION: single cells
6.1. add more 9-45 mL of 2x TrypLETM, transfer tubes to a Tube rotator and place in an incubator  10 rpm, 10 min, 37 °C 
SECTION: single cells
8. discard supernatent and solve pallet in adequat amount of culture medium 1-5 mL 

SECTION: single cells
8.1. filtrate cell solution through a 40 µm Filter to exclude micro carrier fragments
SECTION: Yield, viability and follow up
9. count cells and determine viability 
SECTION: sampling
1.1. take a sample from each tube for counting to determine available cell number
SECTION: Yield, viability and follow up
10. use cells for further analysis
TITLE: Protein isolation from the extract of coelomic fluid of the Lumbricus terrestris.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. Six earthworms, Lumbricus terrestris, after removal from the earth are placed in a 15 ml glass tube and washed thoroughly with PBS, pH 7.4.
?. The body of the annelid is pierced with an 18-gauge needle.
?. The coelomic fluid content of the annelid is added to 2 ml of PBS, pH 7.4.
?. After incubation at 4oC for 1h, 0.9 ml chloroform is added.
?. The mixture is centrifuged at 1000 x g for 5 min at 4oC.
?. The supernatant (1.1 ml) is collected and equal volume of cold ethanol is added drop wise to the preparation.
?. The mixture is then incubated at 4oC for 12 h.
?. Then, it is centrifuged at 4oC for 5 min.
?. The pellet (protein extract) is resuspended in 0.3 ml of PBS, pH 7.4.
?. The preparation is dialyzed against 1L of PBS, pH 7.4 for 12 h at 4oC.
?. The protein concentration is assessed by the Bradford method or any available method.
TITLE: Test with subprotocols
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a test.</div></div>

[STEPS]
?. [Day 1: Sample Prep]
This section has instructions for sample processing and preparation.
?. [Day 2: Culturing]
valuesvalues * 215=A2*217=A3*219=A4*2
valuesvalues * 215=A2*217=A3*219=A4*2
?. [Day 2: Culturing]
TITLE: T cell cytotoxicity (xCELLigence platform)
AUTHORS: Kristin Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps for preparing cellular cytotoxicity assays using the xCELLigence platform.</div><div class = "text-block">In brief: For impedance assays, 10e5 tumor cells were plated overnight in 100mL media in plastic 96 well VIEW-E plates (ACEA) and grown to confluence (measured using the cell index readout on xCELLigence MP platform, RTCA software, ACEA). Transduced T cells were added at varying E:T ratios (as indicated), and cell index was monitored for up to 5 days. Impedance data was analyzed using xIMT software (v1.2, ACEA).   </div></div>

[STEPS]
?. Add 50ul media to each well of the E-VIEW plate (use 'inverse pipetting' technique to avoid bubbles in the wells), place the plate in the xCELLigence MP cradle, and run the setup step of the xCELLigence software to confirm all the wells and connections on the cradle are functioning properly.
?. Resuspend adherent tumor target cells at 10e5 cells/50ul T cell media.
?. Remove the plate from the cradle and plate 10e5 adherent tumor cells (target cells) to grow overnight in (100uL total volume per well) in plastic 96 well VIEW-E plates. Use 'inverse pipetting' technique to avoid bubbles in the wells. Let plate sit in the hood for 30 minutes for cells to settle. Return each plate to the xCELLigence cradle. Begin recording impedance values at desired time interval.
?. Grow cells to confluence (determine confluence using cell index readout on the xCELLigence MP platform using RTCA data acquisition software).
?. Prepare effector cells at desired concentrations for assy. Pause impedance readings and remove the plate from the cradle. Add transduced T cells, cytolysis control, or media only for growth control, in 50ul volume, to each well (use 'inverse pipetting' technique to avoid bubbles in the wells).
?. Return E-VIEW plate to the xCELLigence cradle and resume recording impedance values until desired stopping point. If running an assay for more than 2-3 days, pause the instrument, remove the plates and add additional media (25-50ul) so that the wells do not dry out.
?. Use RTCA and xIMT software for analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Samantha Coy (srose16@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>De-MetaST-BLAST, written in C++, is a script that can validate degenerate primers by performing in silico PCR amplification of the primers against metagenomic and metatranscriptomic databases (e.g. CAMERA; MG-RAST). The output is a list of in-silco PCR amplicons that are then blasted against GenBank to determine the most homologous sequence to the amplicon.</p>
<p> </p>
<p>Modified from Gulvik, C. A., Effler, T. C., Wilhelm, S. W., &amp; Buchan, A. (2012). De-MetaST-BLAST: A Tool for the Validation of Degenerate Primer Sets and Data Mining of Publicly Available Metagenomes. <em>PLoS ONE,</em><em>7</em>(11). doi:10.1371/journal.pone.0050362</p>
</div>
</div>
</div>

[BEFORE_START]
<p>Requirements:</p>
<p>- Potential degenerate primers</p>
<p>- Linux terminal</p>
<p>- Basic knowledge of navigating directories by command line</p>
<p>- De-MetaST-BLAST program installed</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Molecular Dynamics Simulations
AUTHORS: Annan SI Cook, Shanlin Rao

[DESCRIPTION]
This protocol details molecular dynamics simulations of PI3KC3-C1 on a lipid membrane.

[STEPS]
SECTION: Model Preparation
1. Prepare all-atom structural models of PI3KC3-C1 based on the active conformation with missing regions predicted by AlphaFold-Multimer 2.2.
SECTION: Model Preparation
2. Assign protonation states to amino acid side chains based on pKa predictions by PROPKA.
SECTION: Parameterize ATP
3. Parameterize a molecule of ATP in the VPS34 active site using CHARMM-GUI.
SECTION: Membrane Construction
4. Construct lipid membranes consisting of DOPC (60%), DOPE (20%), DOPS (5%), and POPI (15%) using the insane method.
SECTION: Membrane Construction
5. Randomly distribute coarse-grained lipids in the membrane.
SECTION: Solvation and Equilibration
6. Solvate each membrane patch with 150 millimolar (mM) aqueous NaCl.
SECTION: Solvation and Equilibration
7. Perform an initial equilibration of 200 ns.
SECTION: Conversion to Atomistic Representation
8. Convert the equilibrated membrane patch into an atomistic representation using the CG2AT2 tool.
SECTION: Protein-Membrane System Setup
9. Place atomistic PI3KC3-C1 above the resulting membranes with a minimum distance of ~2 nm between protein and lipid atoms.
SECTION: Further Equilibration
10. Subject the protein-membrane systems to an additional 10 ns of equilibration.
SECTION: Further Equilibration
11. Apply harmonic positional restraints (force constant: 1000 kJ mol⁻¹ nm⁻²) to non-hydrogen protein atoms during this equilibration phase.
SECTION: Production Runs
12. Perform 2 µs production runs for each of the six independent replicates of the simulation system.
SECTION: Production Runs
13. Maintain system pressure at   and temperature at 310 °К using the Parrinello-Rahman barostat and velocity-rescaling thermostat.

SECTION: Electrostatic Interactions
14. Treat long-range electrostatic interactions using the smooth particle mesh Ewald method with charge interpolation through fourth-order B-splines.
SECTION: Integration Time Step
15. Use a 2 fs integration time step for the simulations.
SECTION: Bond Constraints
16. Apply the LINCS algorithm to constrain covalent bonds involving hydrogen atoms.
SECTION: Steered Molecular Dynamics (SMD)
17. For SMD simulations, apply harmonic restraints (force constant: 100 kJ mol⁻¹) to reduce the center-of-mass z-distance between the protein group of interest and the membrane lipids underneath.
SECTION: Steered Molecular Dynamics (SMD)
18. Apply a negative rate of -0.5 nm ns⁻¹ until the protein group reaches the membrane surface.
SECTION: Relaxation
19. Allow the complex to relax over periods of 2 µs during further simulations upon removal of any steering force.
TITLE: Protocol for DNA Extraction from Tepary Bean
AUTHORS: Aparna Srinivasan, Magdalena M Julkowska

[DESCRIPTION]
Protocol to extract high quality DNA from Tepary Bean leaves using modified CTAB method.

[STEPS]
SECTION: Collection of leaf sample
1. 
Collect 100 mg of young leaf from Tepary Bean plants in a 2ml eppendorf tubes containing two sterile glass beads and freeze immediately in liquid nitrogen.
SECTION: DNA Extraction
4. 
Add equal volume of Phenol: Chloroform (phenol:chloroform kit), and vortex well.
   

Centrifuge at 14000 rpm for 15 min. Transfer the upper aqueous layer to a new 1.5 ml Ep tube.
  

Do not touch below layers while transferring the aqueous phase. 

Repeat the Phenol: Chloroform alcohol step if the aqueous phase is not clear.

SECTION: DNA Extraction
5. 
Add equal volume of isopropanol, and mix very gently, keep for few minutes to precipitate nucleic acid.
   
Centrifuge at 14000 rpm for 10 min. 

Pellet can be seen at the bottom. 

Discard the supernatant.
SECTION: DNA Extraction
3. 
After 30 min, remove the tubes from water bath and centrifuge at 14000 rpm for 15 min. 

Transfer cleared lysate of 500 μl to a new 1.5 ml tube. Add RNase (10 mg/ml) and keep at 37 °C for 30 min.
SECTION: DNA Extraction
6. 
Add 300 μl of 3M Sodium Acetate and 500 μl 70 % ethanol and centrifuge at 14000 rpm for 5 min.

Discard the supernatant.
SECTION: DNA Extraction
7. 
Add 200 μl 70 % Ethanol and centrifuge at 14000 rpm for 5 min.

Discard the supernatant.
SECTION: DNA Extraction
8. 
Air-dry the pellet, dissolve the pellet in 50 μl TE Buffer.

SECTION: DNA Extraction
2. 
Add 600 μl of pre-warmed CTAB buffer and proteinase K (20 mg/ml), invert the tubes gently to mix together and incubate the tubes in water bath at 65 °C for 30 min. 

Invert the tubes once in every 10 min to homogenize the tissue and buffer.

 
9. 
DNA is run on a 0.8% Agarose gel to check whether it is degraded or having RNA contamination. 

If RNA is present, treat the samples with RNAse A and precipitate again from phenol:chloroform step 4. 

Nucleic acid concentration is measured in Nanodrop, and Ratio of A260/A230 with 1.8-2.0 indicates purity of DNA.

In case the values are <1.8, it indicates contamination such as carbohydrate or phenol.

 
10. 
Store DNA sample at -80 °C until use.
TITLE: Whole-cell patch-clamp in vitro
AUTHORS: Nicola Biagio Mercuri

[DESCRIPTION]
Evoked firing of single neurons

[STEPS]
SECTION: Patch-clamp recordings
1. A single slice is transferred to a submerged recording chamber (2.5–3 ml/min, 33.5°C), on the stage
of an upright microscope equipped for infrared video microscopy (Hamamatsu, Tokyo), allowing direct visualization of the recorded neurons.
SECTION: Patch-clamp recordings
2. Neurons were recorded in whole cell patch-clamp configuration using 1.5-mm borosilicate glass electrodes (3–4 MOhm), pulled with a vertical puller (PP-83 Narishige) and filled with (in mM): K-gluconate (135), KCl (10), HEPES (10), MgCl2(2), EGTA (0.1), CaCl2 (0.05), Mg2-ATP (4), Na3-GTP (0.3), Phosphocreatine-Na2 (10), adjusted to pH 7.3 with KOH. Membrane currents were
recorded in voltage-clamp mode at a holding potential of -60 mV, using a differential amplifier (Multiclamp 700B, Molecular Devices, LLC., 3860 N First Street San Jose, CA 95134 USA). Signals were filtered at 3 kHz, and digitized at 10 kHz.
SECTION: Patch-clamp recordings
1.1. ACSF composition is (in mM): 126 NaCl, 2.5 KCl,1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3. The solution is saturated with a mixture of O2/CO2
TITLE: Combinatorial selective ER-phagy remodels the ER during neurogenesis
AUTHORS: Melissa Hoyer, Cristina Capitanio, Ian R. Smith, Julia C. Paoli, Anna Bieber, Yizhi Jiang, Joao A. Paulo, Florian Wilfling, Brenda A. Schulman, Harper JW

[DESCRIPTION]
The endoplasmic reticulum (ER) employs a diverse proteome landscape
to orchestrate many cellular functions, ranging from protein and lipid
synthesis to calcium ion flux and inter-organelle communication. A case
in point concerns the process of neurogenesis, where a refined tubular
ER network is assembled via ER shaping proteins into the newly formed
neuronal projections to create highly polarized dendrites and axons.
Previous studies have suggested a role for autophagy in ER remodelling,
as autophagy-deficient neurons in vivo display axonal ER accumulation
within synaptic boutons, and the membrane-embedded ER-phagy receptor
FAM134B has been genetically linked with human sensory and autonomic
neuropathy. However, our understanding of the mechanisms underlying
selective removal of the ER and the role of individual ER-phagy receptors is
limited. Here we combine a genetically tractable induced neuron (iNeuron)
system for monitoring ER remodelling during in vitro differentiation with
proteomic and computational tools to create a quantitative landscape of ER
proteome remodelling via selective autophagy. Through analysis of single
and combinatorial ER-phagy receptor mutants, we delineate the extent to
which each receptor contributes to both the magnitude and selectivity of
ER protein clearance. We define specific subsets of ER membrane or lumenal
proteins as preferred clients for distinct receptors. Using spatial sensors and
flux reporters, we demonstrate receptor-specific autophagic capture of ER
in axons, and directly visualize tubular ER membranes within
autophagosomes in neuronal projections by cryo-electron tomography. This molecular
inventory of ER proteome remodelling and versatile genetic toolkit provide
a quantitative framework for understanding the contributions of individual
ER-phagy receptors for reshaping ER during cell state transitions.



[STEPS]
TITLE: Single-Cell Isolation of Human Articular Cartilage
AUTHORS: hswahn  , Martin  Lotz

[DESCRIPTION]
This is a protocol that describes the process of isolating single cells from healthy knee cartilage for single cell RNAseq.

[STEPS]
1. ~1.5 g of articular cartilage from healthy donor knees (grades 0-1) is resected from the medial condyle of the proximal femur. For details regarding the tissue harvesting procedure please see 
dx.doi.org/10.17504/protocols.io.14egn7996v5d/v1.
2. Cartilage shavings are washed with Room temperature Dulbecco's Phosphate Buffered Solution (DPBS) supplemented with 10% calf serum (CS), 1% Antibiotic : Antimycotic (Anti-Anti) and 1% Penicillin-Streptomycin-Glutamine (PSG).

3. Cartilage shavings are minced with a #21 Feather disposable scalpel, and digested in 20mL Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% Anti-Anti and 2% collagenase type II with 100 rpm shaking at 37 °C for 120 min.

4. Cells are gently passed through a 100 µm filter into a 50 mL centrifuge tube followed by gentle passage through a 40 µm filter into a fresh 50 mL centrifuge tube.
5. Filtered cells are spun down at 1200 rpm for 5 minat 37 Room temperature

6. The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10mL of Room temperature DPBS supplemented with 5% CS and 5 mM Ethylenediamine tetraacetic acid (EDTA).

7. Resuspended single cells are treated with DNase I at a concentration of 100 ug/mL for 15 min at 37 Room temperature.

8. Single cells are spun down at 1200 rpm for 5 min at 37 Room temperature

9. The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10 mL of DPBS supplemented with 0.04% Bovine Serum Albumin (BSA).
10. The Invitrogen Countess II FL automated cell counter is used to quantify single cells and determine cell viability. Live cells are determined by trypan blue staining. If >70% cell viability is confirmed, the single cell suspension is diluted to a concentration of 1x106cells/mL for single cell RNA-seq library preparation.
TITLE: Microsphere Synthesis 
AUTHORS: Matthew Woodworth

[DESCRIPTION]
Microspheres are micron- sized plastic beads that have been used in drug delivery systems, typically with poly-lactide:glycolide (PLGA). The PLGA beads are able to encapsulate small molecules and they will degrade over time, releasing the drug. Synthesis of microspheres involves Water-in-Oil synthesis; PLGA or PLA is dissolved in Dicloromethane (DCM), creating the organic phase, and it is added drop-wise to an emulsifier (PVA), the aqueous phase, that is being mixed at a high rate of speed. The consistency of the beads and the size of the beads are dependent on mixing speed. This protocol aims to create PLA microspheres in order to create 'Mock organoids' utilizing the Water-in-Oil' synthesis methodology.

[BEFORE_START]
Collect all materials, and read SDS.

[STEPS]
SECTION: Dissolving PLA
2. Depending on the amount weighted, calculate the amount of DCM needed. The final concentration of PLA in DCM is 50 mg/mL 
SECTION: Dissolving PLA
1. Collect PLA and record the weight. Aim for 0.5-1 gram. 
SECTION: Dissolving PLA
3. In either a glass scintillation vial or a glass pyrex bottle, add the PLA to the DCM. DCM dissolves plastic, do not allow it to come in contact with the plastic lid of the scintillation vial or the pyrex bottle. Use aluminium foil and parafilm to make a makeshift cap. 
SECTION: Dissolving PLA
4. Wait till the PLA is fully dissolved 
SECTION: Water-In-Oil emulsion
5. Create 50 mL of a 0.5% solution of PVA in water.
SECTION: Water-In-Oil emulsion
6. Add the 0.5% PVA solution to a 250 mL Glass beaker. 
SECTION: Water-In-Oil emulsion
7. Place beaker onto a stirring mantle and add a stir bar to the bottom of the beaker.
SECTION: Water-In-Oil emulsion
8. Set the speed to 50% -100% of the max speed; a vortex must be present. 
SECTION: Water-In-Oil emulsion
9. Add the PLA solution dropwise into the PVA. Allow the solution to stir for 5- 20 minutes. Continue to next step.
SECTION: Microsphere collection
10. Place cell strainer on top of 100 mL or 250 mL erlenmeyer flask. 
SECTION: Microsphere collection
11. Decant the PVA solution containing the microspheres over the 100 micron cell strainer. 
SECTION: Microsphere collection
12. Wash the microspheres with excess water. Collect microspheres in a 50 mL conical tube. 
SECTION: Microsphere collection
13. Add a 0.22 micron filter to a pyrex bottle, and decant the once filtered solution of PVA. 
SECTION: Microsphere collection
14. Label the containers with chemical waste contents.
TITLE: Loop L1 (odd level) type IIS cloning - pCk-ye vectors
AUTHORS: Eftychis Frangedakis, Susana Sauret-Gueto, Anthony West, Nicola Patron, marta tomaselli, Marius Rebmann, Jim Haseloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol based on:</div><div class = "text-block"><a href="http://paperpile.com/b/VyRuHk/Qi6v" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640</span></a><span></span></div><div class = "text-block"><a href="https://doi.org/10.1111/nph.15625" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1111/nph.15625</span></a></div></div>

[STEPS]
?. Determine the concentrations of the DNA parts by spectrophotometry (Nanodrop).
?. . Prepare Loop assembly Level 1 reaction master mix (MM) according to Table, if four or less number of parts are assembled into a pCk-pe vector.   AB1  Components
    Volume (μL)
  2  Sterile water 
    3 
  3  10x T4 ligase buffer (NEB)
    1 
  4  1 mg/mL bovine serum albumin (NEB)
    0.5 
  5  T4 DNA ligase at 400 U/μL (NEB)
    0.25 
  6  10 U/μL BsaI (NEB)
    0.25 
  7  Final volume
    &nbsp;5
AB1  Components
    Volume (μL)
  2  Sterile water 
    3 
  3  10x T4 ligase buffer (NEB)
    1 
  4  1 mg/mL bovine serum albumin (NEB)
    0.5 
  5  T4 DNA ligase at 400 U/μL (NEB)
    0.25 
  6  10 U/μL BsaI (NEB)
    0.25 
  7  Final volume
    &nbsp;5
?. Prepare plasmids mix for each reaction, by adding in a 0.2 mL tube, 1 μL of each DNA part aliquot (see step 2), 1 μL of the pCk-pe vector and sterile water up to 5 μL. Mix well.
?. Add 5 μL of master mix to the 5 μL of plasmids mix, to a final volume of 10 μL. Mix well. If more than 4 DNA parts are to be assembled into a pCk-pe vector, reduce the water volume in the MM by 1 μL for each extra 1 μL of DNA part added in the plasmids mix.
?. Place samples in a thermocycler and use the following program: Assembly: 26 cycles of 37​ °C for 3 min and 16 °C for 4 min. Termination and enzyme denaturation: 50 °C for 5 min and 80 °C for 10 min.
?. Transform 20 μL of chemically competent E. coli cells (transformation efficiency of 1 × 107 transformants/µg plasmid DNA) using 2 μL of the Loop assembly reaction and then plate on LB agar plates containing 50 μg/mL kanamycin.
?. Incubate overnight at 37 °C.
?. Colonies with white color are likely to contain the vector with the insert while yellow color colonies will contain the empty vector.
?. Confirm the presence of the correct insert with Sanger sequencing using the primers  pC_F and pC_R
?. 
?. Prepare aliquots for DNA parts to be assembled at a concentration of 15 nM and of the pCk vector at a concentration of 7.5 nM.To calculate the concentration needed for each part to assemble (not the backbone) in ng/µL, divide the length of the plasmid where they are cloned by 100. For the backbone, divide the plasmid length by 200. In this way you can prepare aliquots of your parts and backbones and add 1 µL of each one to the plasmid mix.
?.