eamag/protocols-with-steps huggingface dataset:
TITLE: Density-based removal of bacteria from a culture of the marine heterotrophic flagellate Cafeteria roenbergensis 
AUTHORS: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Heterotrophic flagellates require co-culture with bacteria that serve as a food source. This protocol explains how to reduce the bacterial background of cultures of </span><span style = "font-style:italic;">Cafeteria roenbergensis.</span></div></div>

[STEPS]
?. [Preparation of Cafeteria roenbergensis culture]
Determine the cell density of aCafeteria roenbergensis culture: stain 10 µL of Cafeteria culture with 1 µL of Lugol’s acid iodine solution and count them on a haemocytometer (Neubauer Chamber)
?. [Preparation of Cafeteria roenbergensis culture]
Resuspend the cell pellet in 900 µL of 1X Gradient Buffer (0.5M NaCl, 1x PBS).
Pipette several times to break up cellular aggregates
?. [Preparation of Cafeteria roenbergensis culture]
Centrifuge the Cafeteria cells for 5 min at 4,500 g, 20°C
If it is possible use 50 mL Falcon-types to reduce cell loss
?. [Preparation of Cafeteria roenbergensis culture]
Dilute the Cafeteria culture to 5 x 105 cells/mL in f/2 medium + 0.03% yeast extract and let them grow O/N at 20-25°C
?. [Preparation of OptiPrep density gradient]
Under-layer the cell suspensions approx. 4 ml of 10% solution of OptiPrep using a syringe and a flat needle.
The OptiPrep stock should be diluted in 1X Gradient Buffer
?. [Preparation of OptiPrep density gradient]
Load approx. 4 ml of 20% solution of Optiprep  underneath the 10% solution using a syringe and a flat needle.
The OptiPrep stock should be diluted in 1X Gradient Buffer
?. [Ultracentrifugation]
Centrifuge the tubes using an ultra-centrifuge at  20000 rpm, 20°C for 20 min with slow braking.
?. [Cell recovering]
Recover Cafeteria cells from the gradient, at the interphase between the 10% and 20% Optiprep layers. You can collect them by pipetting from the top or use a syringe and needle.
?. [Cell recovering]
Checked the samples under the light microscope for the presence of bacteria and to determine flagellate density.
It is easier to dilute the collected samples in at least 10 ml of fresh f/2 media, before observing the cells. Depending on the bacterial populations, separation can not be as effective. You can use a gradient of OptiPrep (from 30 to 10%) to determine better conditions.
?. [Cell recovering]
C. roenbergensis can be left in f/2 medium (no added yeast extract) overnight without affecting flagellate viability
Account for about 30% of cell loss from the initial stock (from step 4)
?. [Preparation of OptiPrep density gradient]
In SW40 Ultra-Clear centrifuge tubes, load not more than 5 ml of the cells suspension in 1X Gradient Buffer.
?. [Cell recovering]
It is recommended to remove the OptiPrep from the flagellates by centrifuging them at 4,500 g, 5 min, room temperature.
TITLE: immunehistochemistry and transcriptomic analysis of iodine and breast cancer
AUTHORS: Carmen  Aceves

[DESCRIPTION]
The present work analyzes the antineoplastic effects of molecular iodine alone and in combination with the neoadjuvant therapy FEC/TE (5-fluorouracil/epirubicin/cyclophosphamide or taxotere/epirubicin) in women with breast cancer. In this protocol the immunohistochemistry methodology for the quantification of the estrogen receptor and the proteins associated with the installation of the mesenchymal epithelium transition (e-cadherin and vimentin), immune response (CD8) and cell death (TUNEL) is described, and the methodology for transcriptomic analyses includes: RNA-Seq and transcriptomic analysis; pathway enrichment analysis; validation of the immune response and epithelium/stroma rate and, prediction of the transcription factor regulatory network and peroxisome proliferator-activated receptors type gamma (PPARg) interaction

[STEPS]
TITLE: Tissue processing and freezing after surgery
AUTHORS: Bettina Ergün

[DESCRIPTION]
The aim of this protocol is to document the processing of fresh tissue after surgery.
 
The collected tissue must also be documented in the files which are linked down below
 
The protocol descibes in detail how to process the resulting tissue from following protocols:
 

 






[STEPS]
SECTION: Sample processing in the lab
1. 
Fresh tissue should be processed on the same day if possible, but can be stored at 4°C for a maximum of overnight.
To reduce pathogens, the tissue should be incubated for 60 min atRoom temperature 

SECTION: Sample processing in the lab
2. 
 
 

 

SECTION: Sample processing in the lab
3. 
 

SECTION: Sample processing in the lab
4. 

The tissue is divided in the lab into pieces with an edge length of 1-2 mm.
Mix the fragments of a tumor piece so that each aliquot is as representative as possible of the tumor area.
SECTION: Sample processing in the lab
5. 16 (decision from June 29, 2023; previously: 8) fragments are stored in 1 ml of freezing medium in a cryotube.
If there are enough fragments for more than 4 cryotubes, more fragments can be combined in one tube. A maximum of 5 cryotubes of each entity are frozen.

Cryo tubes should be labelled as follows and documented in the N2 binder:
Tissue ID,T1/T2 resp. N1/N2...
No. of Pieces
Date
SECTION: Sample processing in the lab
6. Freeze samples in Mr. Frosty overnight.
SECTION: Documentation
7. Store patient data and enter the corresponding liquid number:
https://charitede.sharepoint.com/:x:/r/sites/UroForsch/_layouts/15/doc2.aspx?sourcedoc=%7B1282209D-5A7C-4998-A577-145386116E54%7D&file=Biobank_Gewebe_ab01012019.xlsx&action=default&mobileredirect=true
SECTION: Documentation
8. Enter the corresponding tissue number in the liquid collection list:
https://charitede.sharepoint.com/:x:/r/sites/UroForsch/_layouts/15/Doc.aspx?sourcedoc=%7BCE8CD395-BFB0-4B2C-8220-37FB802726A7%7D&file=Biobanking-lfdListe_ab190311.xlsx&action=default&mobileredirect=true
SECTION: Documentation
9. Please add pictures of:
the orgin of the sample in the organ (if avaible)
each tissue piece that has been taken before cutting the fragments. The picture should include the tissue ID (in the picture)
the documentation sheet
TITLE: RNA-Seq Data Analysis (Bowtie-TopHat-Cufflinks)
AUTHORS: Kiichi Hirota

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Library preparation]
Total RNA was extracted from cells using RNeasy® Mini Kit (Qiagen).
?. [Library preparation]
Poly(A) RNA libraries were constructed using TruSeq® Stranded mRNA Library Prep Kit (Illumina).
?. [Sequencing]
The libraries were sequenced at 100 bp paired-ends on an Illumina HiSeq 2500 platform.
?. [Sequencing]
Sequencing data in FASTQ format were deposited in the DDBJ Sequence Read Archive.
?. [Data analysis]
The quality of sequence data was evaluated by FastQC after the trimming process by fastx_toolkit.
?. [Data analysis]
The human reference sequence file (hs37d5.fa) was downloaded from the 1000 genome ftp site (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/), and the annotated general feature format (gff) file was downloaded from the Illumina iGenome ftp site (ftp:// igenome:G3nom3s4u@ussd-ftp.illumina.com/Homo_sapiens/NCBI/build37.2/).
?. [Data analysis]
The human genome index was constructed with bowtie-build in Bowtie.
?. [Data analysis]
The fastq files were aligned to the reference genomic sequence by TopHat with default parameters. Bowtie2 and Samtools was used with the TopHat program.
?. [Data analysis]
Estimation of transcript abundance was calculated, and the count values were normalized to the upper quartile of the fragments per kilobase of transcript per million fragments mapped reads (FPKM) using Cufflinks (cuffdiff).
?. [Data analysis]
Metascape was used for the gene set enrichment analysis. A gene list for metascape analysis was generated using the output from the cuffdiff program.
?. [Data analysis]
Gene ontology annotations were extracted using Ensembl Biomart and sorted by corresponding values (common logarithms of ([FPKM of RCC4-EV] + 1)/([FPKM of RCC4-VHL] + 1)) calculated from the same cuffdiff output file.
?. [Data analysis]
The integer one was added to all FPKM values because we cannot calculate the logarithm of 0. The histogram was generated using TIBCO Spotfire Desktop.
TITLE: UC Davis - Genotyping
AUTHORS: Brandon Willis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Standard PCR conditions for genotyping of mutant mouse DNA extracted from somatic (e.g., tail) tissue.</div></div>

[STEPS]
?. I. DNA Extraction using DNEasy kit.Procedure   1. Make a cocktail of ATL with 180uL buffer ATL and 20uL Proteinase K. Add 200uL to each tube. Be sure tail snip is submerged. 2. Incubate at 55°C for 6-12 hours in heat block or water bath.  3. Remove tubes from heat source and vortex.  4. Add 400 ul of AL/Ethanol mixture to each tube. Vortex.5. Pour liquid into an appropriately labeled Qiagen spin column. Centrifuge at 8,000 rpm for 1 minute. NoteAL/Ethanol mixture is 1 part AL buffer to an equal part of Ethanol. Usually made 50 ml at a time, with 25 ml of AL and 25 ml of 200 proof (100%) Ethanol.  6. Transfer spin column to a new collection tube and add 500 ul of AW1. Be sure ethanol (200 proof) has been added to AW1 buffer; if you add ethanol to AW buffer, be sure to mark the bottle. Spin at 8,000 rpm for 1 minute. 7. Transfer spin column to new collection tube. Add 500 ul of AW2. Again, be sure 200 proof ethanol has been added. Spin at max speed (14,000 rpm) for 3 minutes. 8. Carefully remove tubes from centrifuge. Transfer to a 1.5 ml microtube. Check each spin column for Ethanol before placing in the microtube! If ethanol is still on the spin column, pour off the fluid from the collection tube and spin again for 1 minute at 14,000 rpm. Residual ethanol may inhibit PCR! 9. Add 200 ul of Buffer AE (elution buffer) to spin columns in microcentrifuge tubes. Incubate at room temp for 2 minutes. Spin at 8,000 rpm for 1 minute. 10. Once you have eluted twice (for a total of 200 ul in each tube), label each tube with the sample info that was put on the spin column. Be sure to date the first, last, and approximately every fifth tube for each batch of tails. Store in a cardboard box in the 4oC refrigerator for short term and -20oC for long term.Warning: use appropriate PPE at all times including lab coat, gloves, and eye protection.
?. II. PCR1. Prepare the following master mix on ice:2. Briefly vortex master mix and transfer 18.8 uL of to each 200ul thin walled reaction tube on ice.  3. Briefly vortex DNA and input 1.2 uL of ~100 ng DNA into reaction tube. 4. PCR with the following thermal conditions
?. III. Data Analysis.1. Prepare a 1.2% TBE Agarose gel (~1min per 100ml) with 10ul SyberSafe per 100ml agarose.  2. Line pipette tips with Loading Dye and mix with finished PCR reaction. 3. Inject 75% reaction volume into well (15 ul).4. Run gel at 120 volts for 1.5 hours in TBE.  TBE just covering the gel. 5. Image under UV and adjust and store image for record. 6. Controls: non-template control, isogenic wild type, 1kb plus ladder.  (positive control if available).Warning* where UV protection during imaging.
TITLE: PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Please note that protocols with </span><span style = "font-style:italic;">Q5U Hot Start High-Fidelity DNA Polymerase</span><span> may differ from protocols with other polymerases. Conditions recommended in the protocols should be used for optimal performance.</span></div></div>

[STEPS]
TITLE: Glass Milk preparation - forked
AUTHORS: Martin Codyre

[STEPS]
?. [Glass Milk Preparation]
To prepare glass milk, 325 mesh silicon dioxide (Spectrum Chemicals - SI108) was combined with an excess volume of 10% HCl (~3 N HCl) made from combining 37%and MilliQ water (Millipore) in a fume hood
(dry silica powder should not be inhaled).
?. [Glass Milk Preparation]
After acid washing for   at room temperature  , silica was pelleted by spinning two minutes at  5,000 xg and the supernatant was poured off.
[Possibly 4 to 8 hours]
0 Room temperature
Centrifuge: 5000 33, 2 min
?. [Glass Milk Preparation]
The pellet was resuspended in four pellet volumes of  and then pelleted again.This wash step was repeated for a total of six washes.
?. [Glass Milk Preparation]
The pellet was then washed with four pellet volumes of 10 mM Tris HCl, pH = 8 (ThermoFisher Scientific AM9855G) and 1 mM EDTA (ThermoFisher Scientific 15575020), and pelleted.
?. [Glass Milk Preparation]
Finally, the pellet was resuspended in 1 pellet volume of 10 mM Tris HCl and 1 mM EDTA and autoclaved. This autoclave step is likely superfluous, however, as acid washes should render the beads free of contaminants. The resulting 50% glass milk slurry can be stored at room temperature. Before use, care must be taken to vigorously resuspend the particles as they begin to settle quickly.
?. [Glass Milk Preparation]
Sub step in forked version for testing.
TITLE: Rapid Diagnostic Tests and ELISA for diagnosing Chronic Chagas Disease: Systematic revision and meta-analysis
AUTHORS: Sandra Helena Suescún-Carrero, Philippe Tadger, Carolina Sandoval Cuellar, lauramirez, Lluis Armadans-Gil

[DESCRIPTION]
Objective
To determine the diagnostic validity of the enzyme-linked immunosorbent assay (ELISA) and Rapid Diagnostic Tests (RDT) among individuals with suspected chronic Chagas Disease (CD). 

Methodology 
A search was made for studies with ELISA and RDT assays validity estimates as eligibility criteria, published between May and August 2020 on PubMed, Web of Science, Scopus, and LILACS. This way, we extracted the data and assessed the risk of bias and applicability of the studies using the QUADAS-2 tool. The bivariate random effects model was also used to estimate the overall sensitivity and specificity through forest-plots, ROC space, and we visually assessed the heterogeneity between studies. Meta-regressions were made using subgroup analysis. We used Deeks’ test to assess the risk of publication bias.

Results
43 studies were included; 27 assessed ELISA tests; 14 assessed RDTs; and 2 assessed ELISA and RDTs, against different reference standards. 51.2 % of them used a non-comparative observational design, and 46.5 % a comparative clinical design (“case-control” type). High risk of bias was detected for patient screening and reference standard. The ELISA tests had a sensitivity of 99% (95% CI: 98-99) and a specificity of 98% (95% CI: 97-99); whereas the Rapid Diagnostic Tests (RDT) had values of 95% (95% CI: 94-97) and 97% (95% CI: 96-98), respectively. Deeks’ test showed asymmetry on the ELISA assays.


Conclusions
ELISA and RDT tests have high validity for diagnosing chronic Chagas disease. The analysis of these two types of evidence in this systematic review and meta-analysis constitutes an input for their use. The limitations included the difficulty in extracting data due to the lack of information in the articles, and the comparative clinical-type design of some studies. This article was funded through the Universidad de Boyacá. 

[STEPS]
1. Protocol and registration
2. Eligibility criteria
3. Data sources
4. Study search and selection
5. Data collection process
6. Definition for data extraction
7. Risk of bias and applicability
8. Diagnostic accuracy measures
9. Summary of results
10. Addinitional analyses
11.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Materials:</strong><br /><br />1) SOB medium</p>
<ul>
<li>1.0% Bacto-tryptone</li>
<li>0.5% Bacto-yeast extract</li>
<li>10.0 mM NaCl</li>
<li>2.5 mM KCl</li>
<li>10.0 mM MgCl<sub>2</sub></li>
<li>10.0 mM MgSO<sub>4</sub></li>
<li>Prepare the MgCl<sub>2</sub> and MgSO<sub>4</sub> as 1 M stock solutions and autoclave separately. </li>
<li>Add the MgCl<sub>2</sub> and MgSO<sub>4</sub> after sterilization of the remainder of the components. </li>
</ul>
<p><span style="font-size: 10.8333px;"> </span></p>
<p>2) SOC medium</p>
<ul>
<li>SOB media, supplemented with 20mM glucose</li>
</ul>
<p>3) TFB buffer</p>
<ul>
<li>10 mM K-MES, pH 6.2, 100 mM KCL, 45 mM MnCl<sub>2</sub>:4H<sub>2</sub>O, 10 mM CaCl<sub>2</sub>:2H<sub>2</sub>O, 3 mM HaCoCl<sub>3</sub></li>
<li>Weigh out the components. Add the MES to d-H<sub>2</sub>O and adjust the pH with KOH.</li>
<li>Add the remaining components (in order), waiting until one component is in solution before adding the next. Adjust the volume to the final volume. Filter sterlize. Store frozen at -20<sup>o</sup>C in 15 mL aliquots. </li>
</ul>
<p>4) DTT solution</p>
<ul>
<li>2.25 M DTT, 40 mM KOAc, pH 6.0. Filter sterilize, Store frozen at -20°C.</li>
</ul>
<p>5) DMSO (Dimethylsulfoxide)<br /><br /><strong>Reference</strong><br /><br />D. Hanahan.  (1983).  Studies on Transformation of <em>Escherichia</em> <em>coli</em> with plasmids.  <u>Journal of Molecular Biology</u> <strong>166</strong>:  557-580.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Methods for TransEpithelial Electrical Resistance (TEER) and short circuit current measurement of the effects of soluble mediator on intestinal epithelial cells. </p>

[BEFORE_START]
<p>Prepare Forskolin (10µM), UTP (100µM), Amiloride (10µM), DIDS (100µM) and GlyH-101 (50 µM) in aqueous solution and Amiloride (10mM), GlyH-101 (50mM) in DMSO.</p>

[GUIDELINES]
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a modified protocol from the original NEB splitting the 50uL aliquot into two separate tubes for dual reactions from a single tube.

[GUIDELINES]
<strong>Transformation Protocol Variables</strong><br /><br /><strong>Thawing</strong>: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.<br /><br /><strong>Incubation of DNA with Cells on Ice</strong>: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.<br /><br /><strong>Heat Shock</strong>: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.<br /><br /><strong>Outgrowth</strong>: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.<br /><br /><strong>Plating</strong>: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.<br /><br /><strong>DNA Contaminants to Avoid</strong><br />
<table width="468">
<tbody>
<tr>
<td><strong>Contaminant</strong></td>
<td><strong>Removal Method</strong></td>
</tr>
<tr>
<td>Detergents</td>
<td>Ethanol precipitate</td>
</tr>
<tr>
<td>Phenol</td>
<td>Extract with chloroform and ethanol precipitate</td>
</tr>
<tr>
<td>Ethanol or Isopropanol</td>
<td>Dry pellet before resuspending</td>
</tr>
<tr>
<td>PEG*</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
<tr>
<td>DNA binding proteins (e.g. Ligase)</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
</tbody>
</table>
<br />* Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Seawater is collected and preserved to be analyzed in lab using an InFlux Flow Cytometer with the Spigot software package. The populations targeted for enumeration are <em>Prochlorococcus</em>, <em>Synechococcus</em>, unclassied pico-eukaryotes (cells sized around 1μm), and heterotrophic non-fluorescing bacteria. Each sample is divided in two, with one aliquot analyzed for autofluorescence and the other for heterotrophic bacteria stained with SYBR Green I. In both cases, excitation is produced using stacked 488nm and 457nm lasers. The data is analyzed using FlowJo (v10) to determine cell concentrations (cells/mL).</p>

[STEPS]
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TITLE: CoVIDScandx
AUTHORS: Muhammad Munir

[STEPS]
?. Add smart lysis buffer with 1:1 ratio in the VTM sample
?. Heat for 68C for 8 minutes and then 95C for 10 minutes
?. Cool and take 3 ul to colorimetric LAMP master mix (NB: M1800)
?. Vortex reaction mix and centrifuge to collect material
?. Pipet 24 μl per reaction into desired reaction vessels and add 1 µl of sample. Mix by vortexing or by pipetting if using a plate or similar vessel, centrifuge to collect if necessary. Check that reaction solutions have a bright pink colour, which indicates initial high pH required for successful pH-LAMP reaction.
?. Incubate at 65°C for 30 minutes.
?. Cool on ice for 2 minutes
?. Add 5 ul of the LAMP reaction with the sample buffer
?. Load to the LFD strip and let is absorbe
?. Visualize the bands for internal control and SARS-CoV-2
?. Scan with mReader to record results