eamag/protocols-with-steps huggingface dataset:
TITLE: ICD-11 post-traumatic stress disorder (PTSD) and complex post-traumatic stress disorder (CPTSD) assessed with the International Trauma Interview (ITI) and International Trauma Questionnaire (ITQ) for trauma-affected civilians and refugees in two outpatient
AUTHORS: Maja Bruhn, Hinuga Sandahl, Mads Christian Jensen, Henriette Laugesen, Sofie Folke, Jessica Carlsson

[DESCRIPTION]
This study protocol outlines a research project aimed at investigating the two assessment tools for post-traumatic stress disorder (PTSD) and complex PTSD (CPTSD) based on the ICD-11 framework: the International Trauma Interview (ITI) and the International Trauma Questionnaire (ITQ). The study is conducted at two specialized outpatient clinics in Denmark, focusing on trauma-affected civilians and refugees.
With a sample size of 100 participants,  equally divided between the two clinics, the study has three primary objectives: (1) to assess the feasibility of implementing the ITI in clinical settings by evaluating clinician time expenditure and clinical utility; (2) to determine the prevalence and diagnostic consistency of ICD-11 PTSD and CPTSD as measured by the ITI and ITQ; and (3) to compare the two study populations on various sociodemographic and clinical variables, including trauma history, mental health symptoms, and functional outcomes. This study employs a mixed-methods approach, integrating quantitative data collection through standardized measures and qualitative insights from clinician focus groups. Results will contribute to the growing body of evidence on PTSD and CPTSD diagnosis, providing insights into the clinical utility of the ITI and the ITQ for diverse trauma-affected populations.

[STEPS]
1.
TITLE: Microorganisms and culture conditions
AUTHORS: Lis Rocha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between four strains of </span><span style = "font-style:italic;">S. aureus</span><span> causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122, especially in genes encoding host immune evasion and surface proteins. The comparison of orthologous genes using OrthoMCL identified a membrane transporter in the genomes of the bacteria belonging to the subclinical group, but this finding was not confirmed by polymerase chain reaction (PCR) on a collection of field isolates of </span><span style = "font-style:italic;">S. aureus</span><span> associated with clinical or subclinical mastits. The secreted and surface proteins predicted by different in silico tools were compared through multidimensional scaling analysis, revealing a high degree of similarity among the six strains. However, differences were seen in the nucleotide sequences of a gene that codes for a hypothetical protein (cl3309) and a lipoprotein (cl3700). These findings were also analyzed by PCR on DNA extracted from field isolates of</span><span style = "font-style:italic;"> S. aureus</span><span>. The lipoprotein, but not the hypothetical protein, was able to separate the clinical isolates from the subclinical ones. These results show that sequence variation among bovine </span><span style = "font-style:italic;">S. aureus</span><span>, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes</span></div></div>

[STEPS]
?. [Growing and storing conditions]
The bacteria used in this study were maintained on BHI agar (Brain Heart Infusion, BHI HiMedia, Mumbai, India) at 37 °C
?. [Growing and storing conditions]
stored in the long term in BHI containing 20% glycerol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This electroporation protocol is for use with the&nbsp;NEB Turbo Electrocompetent E. coli cells (<a href="https://www.neb.com/products/c2986-neb-turbo-electrocompetent-e-coli" target="_blank">C2986</a>). These&nbsp;cells are suitable for high efficiency electroporation and rapid colony growth; they are ideal for DNA library constructions and all cloning purposes.

[STEPS]
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TITLE: JMN-MSMP Muscle RNA Isolation
AUTHORS: ccherry

[DESCRIPTION]
Isolation of RNA from muscle samples

[STEPS]
SECTION: Isolation of RNA
1. Muscle sections were submerged in Trizol Reagent (Thermo Fisher Scientific), minced, and grinded.
SECTION: Isolation of RNA
2. Subsequent RNA isolation was performed using the RNAeasy Mini Kit (Qiagen) following manufacturers protocol.
TITLE: Thawing of hPSCs grown on MEFs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).

General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 
3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.

[STEPS]
1. Prepare one 6-well MEFs plate for each vial of frozen hPSCs
2. Place vial of frozen hPSCs in 37 °C  water bath with constant agitation. 

3. Pipette thawed cell suspension into 10 ml pre-warmed hPSCs medium.


4. Centrifuge  200-300 x g, 5 min 

5. While cells are spinning, aspirate the MEFs medium from the MEFs plates and add 2 ml pre-warmed hPSCs medium + Rock inhibitor to each well. 
 

6. Aspirate most of the medium on the centrifuged hPSCs, being careful not to disturb the pellet
3.1. hPSCs Medium
     DMEM/F12      385 ml        Fetal Bovine   Serum (FBS)      75 ml        Knockout Serum Replacement      25 ml        L-Glutamine (100X)      5 ml        Penicillin & Streptomycin (100X)      5 ml        MEM Non-Essential Amino Acids (100X)      5 ml        2-Mercaptoethanol (10,000X)      50 ul        Heat Stable Recombinant Human FGF2 (25ug/ml)*      80 ul   
 

L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 

Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml   
 
5.1. hPSCs Medium + Rock inhibitor
     hESCs medium      500 ml        Y-27632 (1,000X)      500 ul   
 
Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   
 

7. Add 1 ml hPSCs medium with or without Rock inhibitor (dependent on choice made above).
8. Resuspend the cells using a P1000 tip.
9. Pipette the cells onto the 2 ml already on the MEFs.
10. Check the cells under the microscope to get an idea of the resulting cell density.
11. Spread the cells by moving the plate in left-right, then backward-forward motion.
12. Place the plate in the low oxygen incubator
13. From day 3, change 2-3 ml pre-warmed hPSCs medium for each well daily.
14. When large colonies emerge or hPSCs density reaches 50-70%, passage using collagenase. It usually takes 7-10 days for the thawed cells to grow to this point.
TITLE: Turf Umbrella Runoff Study: Total Suspended Solids (TSS)
AUTHORS: USDA

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Turf Umbrella Runoff Study: Total Suspended Solids (TSS)</div></div>

[STEPS]
?. Remove samples from the refrigerator. Bring to room temperature.
?. Assemble filtering apparatus to vacuum.
?. Record the aluminum tin ID # on data sheet.
?. Using a tweezers, place an oven-baked filter (either side facing up) into the aluminum tin. Discard the blue paper between filters.If you need more baked filters, use a tweezers to place unbaked filters on aluminum foil, making sure that there is layer of aluminum foil between each filter and the next. Seal the filters up and then place in the oven at 104C overnight.
?. Record the weight (0.000X g) of the filter and aluminum tin. Set aside.
?. Record the sample ID and weight (0.0X g) of sample. Be sure to weigh the sample with the cap on to prevent spillage.If you are handling Manifold Water samples, double glove.
?. Using a forceps, seat the filter on the filter holder with either side up. Turn vacuum on. Sparingly wet the filter, with epure water, to create a seal and prevent sample loss.When handling filters, always try and touch the tweezers to only the outer edges of the filter.
?. Carefully place the filtering apparatus top onto the filter holder. Secure with the filtering apparatus clamp.
?. Vigorously shake sample and then add to the filtering apparatus. Slowly filter sample. Remove and discard debris such as leaves, sticks, or fecal matter. Replace the tin foil back on top of the filtering apparatus.
?. After the entire sample has passed through the filter, rinse the inside of the filtering apparatus with epure water 3x.Use a designated squirt bottle for this with fresh MilliQ water (DO NOT use the one that is used for washing dishes as we cannot ensure there aren’t contaminants on it)
?. Record the weight (0.0X g) of the emptied sample bottle with its cap on. Turn off vacuum.
?. Disassemble the filtering apparatus. Using a tweezers, place the filter into the aluminum tin. Rinse the tweezers with MilliQ water between samples. Place in oven at 103 - 105℃ for at least 4 hours, or overnight.Make sure that you take the filter off soon after it’s done filtering. Otherwise it may stick on the filtering apparatus and be difficult to move.If the filter is not coming off, try and wet it with a little MilliQ water to help get it unstuck.
?. Remove tin from oven and desiccate for at least 30 minutes.
?. Record the final weight (0.000X g) of the filter and aluminum tin.
?. Return tin and filter back to oven.
?. Repeat step 13.
?. Repeat step 14.
?. If there is a big difference in weight from step 14 and 17, then rebake and weigh a third time.
TITLE: Transient CRISPR-Cas9 Coupled with Electroporation Protocol
AUTHORS: Amy Gladfelter

[DESCRIPTION]
Transient CRISPR-Cas9 transformation of Cryptococcus neoformans.

[GUIDELINES]
References

1. Lin, X., et al., Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation. Med Mycol, 2015. 53(3): p. 225-234.

2. Fan, Y. and X. Lin, Multiple applications of a transient CRISPR-Cas9 coupled with electroporation (TRACE) system in the Cryptococcus neoformans species complex. Genetics, 2018. 
 
 
 

[STEPS]
1. PCR amplification of CAS9, sgRNA, your construct. 
 
 
CAS9: Use plasmid pXL1-CAS9-HYG as template with primers CAS9-F and CAS9-R. (6985 bp)

sgRNA: U6P and sgRNA scaffold 
 
U6pPromoteris PCR amplified using serotype Dgenomic DNA astemplate with primers U6P-F and GOI-sgRNA-R. ~295 bp 
SgRNA scaffold is PCR amplified using pYF515 astemplate with primers GOI- sgRNA-F and sgRNA-R. ~108 bp 
sgRNA construct is PCR amplified using above two PCR product sastemplate with primers U6P-F and sgRNA-R. ~383 bp 
 
 
 
 
2. Mix2 µg your construct DNA, 100 ng sgRNA, and 170 ng CAS9 in an Eppendorf tube. 
 
 
 

3. Vacuum dry the DNA and elute in5 µLDNAse/RNAse free water.
 


4. Inoculate recipient strain in 5 mL YPD liquid medium, culture overnight at 30 °C with shaking at 250 rpm.
 
 
 
 


5. Use the overnight culture to inoculate100 mLfresh YPD medium at an initial inoculum of OD600=0.2. Grow the cells for additional240 minto300 min until the cell density reached OD600 between 0.6-1.0. 
 From this step on, everything on ice and centrifugation at 4 °C 
 
 
 




6. Collect cells by centrifugation at 3200g for5 minat 4 °C.
 
 
 
 


7.  Wash cells  with ice-cold water (EB Buffer instead of water in 2015 paper).  (wash 1/2)
 
 
 
9. Suspend cells in 10 mLice-cold EB buffer (10 mM Tris-HCl, pH 7.5, 1mM MgCl2, 270 mM Sucrose) with 1mM DTT.
 
 
 
 

10.  Incubate the cells on ice for an hour (60 min).
 



11. (Optional) Wash cells with 10 mLice-cold EB buffer once (2015 paper).
 
 
 
 

12. Collect the cells by centrifugation and resuspend in250 µL EB buffer.
 
 
 
 

13. Mix 45 µL cells with 5 µL DNA mix from step 2  in a pre-cooled 2 mm gap electroporation cuvette.
 
 
 
 



14. Transform the DNA by electroporation using the BioRad gene pulser with settings of 0.45 kV, 125 μF, 600 Ω. (If using an Eppendorf multiporator, use the bacterial mode with V=2 kV with τ optimized for 5 ms)
 
 
 
 
15. Suspend electroporated cells in 1 mLof YPD medium and culture at 30 °C for 120 min (90 minin 2015 paper) before plating onto the appropriate selective agar medium. 
 
 
 




8.  Wash cells  with ice-cold water (EB Buffer instead of water in 2015 paper).  (wash 2/2)
TITLE: In-solution Digestion of ECM-Enriched Proteins Samples for Mass Spectrometry Analysis
AUTHORS: jconsi, Ikram Isa, Alexandra Naba

[DESCRIPTION]

The pellet obtained after the decellularization procedure and removal of SDS is highly enriched in insoluble ECM proteins. For further analysis by mass spectrometry, these proteins need to be digested into peptides. Note that, as a consequence of ECM protein insolubility, it is not possible at this step to measure the protein concentration of the sample. We thus provide volumes of reagents to digest the ECM-enriched samples into peptides based on the size (mm) or dry weight of the ECM-enriched pellet (Table 1). 



[BEFORE_START]
The solutions of ammonium bicarbonate, urea, DTT, iodoacetamide and trifluoroacetic acid all need to be freshly prepared. 
We recommend using low retention microcentrifuge tubes and tips to maximize recovery 

[STEPS]
SECTION: Protein resuspension and reduction
1. Resuspend the ECM-enriched sample by adding the appropriate volume of 8 M urea (urea is dissolved in 100 mM ammonium bicarbonate) to the ECM-enriched pellet and add Dithiothreitol(DTT) at a final concentration of 10 mM (see Table 1). Incubate with continuous agitation at   for 120 min at37 °C.
 

SECTION: Protein alkylation
2. Protein alkylation
SECTION: Protein alkylation
2.1. Prepare a 250mM iodoacetamide solution in 100 mM ammonium bicarbonate.



SECTION: Protein alkylation
2.2. Cool the sample to Room temperatureand add the iodoacetamide to a final concentration of 25 mM. For complete alkylation, the DTT:iodoacetamide ratio should be between 1:2.5 and 1:3.

SECTION: Protein alkylation
2.3. Incubate in the dark for 30 min at Room temperature.

SECTION: Protein deglycosylation
3. Deglycosylation is needed to remove carbohydrate side chains that interfere with identification of peptides modified by N-linked glycosylation.
SECTION: Protein deglycosylation
3.1. Dilute to 2 M urea with 100 mM ammonium bicarbonate and add the appropriate amount of PNGaseF (see Table 1). Incubate with continuous agitation at   for 120 min at 37 °C.

SECTION: Digestion - Day 1
4. Digestion - Day 1
SECTION: Digestion - Day 1
4.1. Add Lys-C (refer to Table 1 for volume amounts) and incubate with continuous agitation at   for 120 minat 37 °C.

SECTION: Digestion - Day 1
4.2. Add trypsin (refer to Table 1 for volume amounts) and incubate with continuous agitation at   120 min at 37 °C 

 

SECTION: Digestion - Day 2
5. Digestion - Day 2
SECTION: Digestion - Day 2
5.1. Add a second aliquot of trypsin (refer to Table 1 for volume amounts)  to the sample and incubate with continuous agitation at   for an additional120 min at 37 °C.

SECTION: Acidification
6. Acidification
SECTION: Acidification
6.1. Upon completion of the digestion, the trypsin is inactivated by acidifying the sample with freshly prepared 50% trifluoro-acetic acid (TFA) in HPLC-grade water. The sample should reach <pH 2 

 
 

SECTION: Acidification
6.2. Centrifuge the acidified sample at 16000 x g for 5 minat Room temperature. Collect the supernatant in a clean low-retention tube. At this point, the peptide solution can be stored at -20 °C prior to desalting: 
 
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TITLE: abcde
AUTHORS: Nicolas Nitsche

[DESCRIPTION]
new test protocol

[STEPS]
SECTION: Day 1
1. add 5 ul buffer 
 

SECTION: Day 1
2. add 5 µL and centrifuge, add   

SECTION: Day 1
3. pipette buffer 

 

SECTION: Day 1
4. step 4

SECTION: Day 1
5. add 5 µL and centrifuge 

SECTION: Day 1
6. pipette buffer 

 

SECTION: Day 1
7. step 4

 

SECTION: Day 1
8. 
SECTION: Day 1
9. 
SECTION: Day 1
10. 
SECTION: Day 1
10.1. 
SECTION: Day 1
10.2. 
SECTION: Day 1
10.3. 
SECTION: Day 1
11. 
SECTION: Day 2
12. add   

SECTION: day 3
13.
TITLE: Immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation
AUTHORS: Mallory Wright, ckremitz, William J Buchser

[DESCRIPTION]
This immunocytochemistry protocol is used for the characterization of IPSC differentiation into motor neurons using several biomarkers: neuroepithelial cells (SOX1), motor neuron progenitors (OLIG2 and NKX2.2), motor neurons (MNX1), and the mature motor neurons (ISL, ChAT, MAP2).

*Primary and secondary antibody information located in materials section
 





[BEFORE_START]
To ensure that the observed results are not just random events, use controls, such as undifferentiated IPSCs, to accurately analyze that the antibodies are working as expected. 

Here is an example of how we seed cells onto a 96-well plate. The 96-well has an equal amount of wells of differentiated (blue) and undifferentiated cells (yellow) for analysis. Cells are also counted and seeded at equal densities.

 


[STEPS]
1. Remove the medium from your cells 
 





2. FIX - Dilute 32% Paraformaldehyde solution to 4% PFA in 1X phosphate-buffered saline (PBS) 
3. Add 100uL of 4% PFA to each well in the 96-well plate. (1ml if using a 6-well plate). Incubate for 15 min at room temperature.

4. Remove the fixative solution and wash with 1XPBS at 100ul per well using a multichannel pipette. Repeat 3 times.
 

5. PERMEABILIZE - Add 100uL of 0.5% Triton X-100 to each well of a 96-well (1ml if using 6-well plate)
6. Incubate for 15 min at room temperature.

7. Remove the permeabilization solution and wash 3 times with 1XPBS 
8. BLOCK - Add 100ul of 3% BSA (bovine serum albumin) or blockAid-blocking solution to each well of a 96-well plate slowly to Block. (1ml if using 6-well plate)
9. Incubate for at least60 min (up to overnight) at room temperature.

10. Calculate the amount of primary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution 
11. PRIMARY ANTIBODIES - Remove 3% BSA from wells and add 100uL of primary antibody per well

 

12. Incubate for 60 min at room temperature in a dark place or overnight at 4°C.    
(Different primary antibody stains may take longer to stain and could require optimization).
13. Remove primary antibody and wash three times slowly with 1xPBS 
 

14. SECONDARY ANTIBODIES - Calculate amount of secondary antibody needed (located in materials section) and dilute in 3% BSA+ .3% TritonX-100 solution and 1:4000 Hoechst. 
15. Add 100uL per well in 96-well and incubate for at least 60 min at room temperature in a dark place.  

16. Remove secondary antibody and wash three times with 1xPBS 
17. Scan on the confocal microscope
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was used for the molecular profiling of the soil fungal community of two winter wheat fields as a part of a long-term field trial in Germany with following Illumina Amplicon Sequencing. Besides different pre-crops, the effects of soil management and fertilization on the fungal communities were investigated.  </p>
<p>Due to different aspects of our protocol, we wanted to ensure reproducing a realistic ratio of the different fungal communities in the soils. The most important steps to conform this demand being the frequently measurements of DNA concentrations at different steps of the protocol, the high numbers of PCRs of each soil sample and the use of the qPCR setting a cut-off for the PCR cycles to avoid an over-amplification of certain PCR-fragments and a loss of detectable biodiversity. The use of the two DNA isolation kits based on the cooperation with our project partner and the exchange of DNAs. </p>

[GUIDELINES]
<p>Gloves sholud be worn at all the time and changed regularly. </p>

[STEPS]
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TITLE:  In vitro germination of Austropuccinia psidii urediniospores 
AUTHORS: Alyssa  M Martino, Rebecca M Degnan

[DESCRIPTION]
Optimisation of Austropuccinia psidii urediniospore germination for use in RNA extraction and cytogenetics. 

[STEPS]
SECTION: Spore collection, desiccation, and storage
2. Move fresh spores to a glass petri dish with no lid. Transfer petri dish to a desiccator with silica gel beads for 24 - 48 hours to dry the spores.


SECTION: Inocula preparation and plating
6. 
In a 15 mL centrifuge tube, make up inocula to a concentration of approximately 1 mg/mL in sterile distilled water (SDW) with 5ul/mL Tween 20 (0.05%). Mix thoroughly by gently inverting the tube several times.
 

SECTION: Incubation
9. Germinate in incubator for a minimum of 8 hours in the dark at 18°C and 75% relative humidity.
SECTION: Determine Germination Percentage
10. For each aliquot, count the number of germinated spores under a dissecting microscope.  A germinated spore is considered to be a spore with a germination tube at least double the length of the urediniospore.
 

SECTION: Inocula preparation and plating
5. Create 2% water agar plates.
SECTION: Inocula preparation and plating
8. Pipette 2 uL aliquots of inocula onto agar plate, covering the entire plate with distinct 2 uL droplets and seal the plates with parafilm.
SECTION: Spore collection, desiccation, and storage
1. Harvest fresh spores from heavily infected leaves by shaking into a paper bag.
SECTION: Spore collection, desiccation, and storage
3. Sieve spores to remove any plant matter or dirt.
SECTION: Spore collection, desiccation, and storage
4. Aliquot spores into Nunc CryoTubes or similar for short or long term storage.

 


SECTION: Inocula preparation and plating
7. Rehydrate spores in inocula for 30 minutes prior to use.