eamag/protocols-with-steps huggingface dataset:
TITLE: Squalene Quantification using Nile Red Staining (Developmental)
AUTHORS: Sebastian Triesch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This protocol is under development!</span></div><div class = "text-block">Nile Red is a fluorescent dye that stains selectively hydrophobic substances. We assume that squalene accumulates in the cell membrane or in lipid vesicles where it can be stained by the dye. </div></div>

[STEPS]
?. [Culture Sampling]
Sample 1-2 ml Synechocystis culture, measure its OD at 750 nm and adjust it to 2 ml of OD (750 nm) = 0.5 in BG-11 media. Split your adjusted culture in 2x 1 ml. One portion will be stained with Nile Red, the other will serve as a negative control.
?. [Staining]
Stain one portion of the previously adjusted culture with c(final) = 5 µg/ml Nile Red in DMSO. Add the same volume of DMSO to the unstained control samples.
Wear gloves and a lab coat when working with DMSO as it stains skin and cloth.
?. [Washing]
Incubate your culures for 60 min in the dark.
[Incubation]
?. [Washing]
Centrifuge all samples for 5 min at 8000 xg.
[Centrifugation]
?. [Washing]
Carefully discard the supernatant by pipetting. Avoid resuspending the pellet or losing cells! Try removing as much supernatant as possible.
?. [Washing]
Thoroughly resuspend all pellets in 1 ml BG-11 media, centrifuge again (5 min at 8000 xg), discard the supernatand and again resuspend the pellet in 1 ml BG-11 media.
[Centrifugation]
?. [Data Gathering]
Fill each 200 µl  of your cultures in 96 well plate's wells. Your sample volume should allow four wells per sample.
?. [Data Gathering]
Measure each well's OD(750 nm) in the plate reader.
?. [Data Gathering]
Measure Nile Red fluorescence. Use following wavelenghts: Excitation: 510 nm/Emission: 660 nm
?. [Data Gathering]
Analyze your data: Normalize each well's fluorescence to the respective OD and subtract fluorescence without Nile Red from fluorescence with Nile Red. Compare your results to wild type Nile Red stained fluorescence.
?. [Washing]
Optional: Keep the removed supernatant to check the quantity of free Nile Red in the supernatant! Ideally, you can repeat the washing steps until no more Nile Red is present in the supernatant.
TITLE: Constant mood monitoring using WhatsApp for persons with depression: a pilot study
AUTHORS: Maritta A Välimäki, Qing Long, Min Yang, Sau Fong

[DESCRIPTION]
Background
Preliminary evidence suggests that social media could provide a convenient platform to monitor mood. It is still unknown whether WhatsApp mood monitoring could be used to identify mood changes in adults. 

Methods
A cohort study design was used in this study. The data is a part of the bigger study where psychological variables together with mobility data were collected using GPS (dx.doi.org/10.17504/protocols.io.5qpvory7bv4o/v1). Participants (N=32) with signs or a diagnosis of depression joined the study. The data were collected between 15 November 2018 and 30 September 2019. The participants were monitored daily for 14 days using WhatsApp. Depressive symptoms, perceived stress, mental well-being, self-esteem, and life satisfaction were collected at baseline and the end of a 14-days. The feasibility of constant monitoring was described as well. 

Results
Although participants’ (N=31) mood status fluctuated, the analysis of ANOVA showed no significant difference in average mood levels during the 14-day period (F=1.985, p=0.093). The paired-sample t-test showed a significant improvement in self-esteem between baseline and follow-up (t=3.978, p<0.001).

Conclusion
WhatsApp could be used for constant mood monitoring as it is easy to use, and it was not found to have a negative impact on participants’ psychological well-being. However, further research with more accurate methods and a larger sample size might be needed to identify patterns in mood changes and their link to well-being. 

[STEPS]
TITLE: Removal of gDNA out of totalRNA
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Start</span></div><div class = "text-block">Calculate the volume required for 1 µg RNA</div><div class = "text-block"><span style = "font-weight:bold;">Digestion of DNA</span></div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Component</td><td rowspan = "" colspan = "" style ="display : table-cell;">amount</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">DNaseI buffer</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 µl</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">total RNA</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 µg</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">DNaseI</td><td rowspan = "" colspan = "" style ="display : table-cell;">1 µl</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">H2O</td><td rowspan = "" colspan = "" style ="display : table-cell;">to 10 µl</td></tr></table><div class = "text-block"><span>pipet components into a RNase-free 1.5 ml tube (following order: H</span><span style = "vertical-align:sub;">2</span><span>O, buffer, total RNA, DNaseI)</span></div><div class = "text-block">incubate reaction at 37°C for 30 min</div><div class = "text-block"><span style = "font-weight:bold;">Extraction of DNaseI-digested RNA</span></div><div class = "text-block">add 1 µl 50 mM EDTA and incubate at 65°C for 10 min</div><div class = "text-block">add 1/10 Volume NaOAc and 3 Volumes 100% EtOH</div><div class = "text-block">freeze at -20°C over night or at -80°C for at least 1h</div><div class = "text-block">centrifuge at 13000 rpm for 10 min at 4°C</div><div class = "text-block">Discard supernatant (RNA pellet could be not visible, important to know where it should be; it is definetly there)</div><div class = "text-block">add 500 µl 70% EtOH</div><div class = "text-block">centrifuge at 13000 rpm for 10 min at 4°C</div><div class = "text-block">discard supernatant, remove as much ethanol as possible</div><div class = "text-block">dry tubes at RT under the hood (~5min), do not overdry!</div><div class = "text-block">resuspend pellet in 20 µl RNase-free water</div><div class = "text-block">measure concentration</div><div class = "text-block"><span style = "font-weight:bold;">Quality control:</span></div><div class = "text-block">perform PCR on your DNA-free RNA (using primers 538/747; 57°C, 653 bp). Include a positive control using gDNA as a template.</div><div class = "text-block">There should be no visible bands! </div></div>

[STEPS]
TITLE: H&amp;E Staining for Pancreas or Eye Cryosections
AUTHORS: Diane  Saunders, Angela  Kruse, Jamie  Allen, Carrie   Romer, Danielle  Gutierrez, Alvin Powers, Jeff  Spraggins

[DESCRIPTION]
This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.

[STEPS]
1. Air dry sections for 5 min .

2. Incubate in 95% alcohol for 1 min , then wash, dipping until clear.

3. Incubate in hematoxylin for 30 s , then wash until clear.

4. Dip 3-4x in bluing solution, then wash for 30 s 

5. Dip 3-4x in 95% alcohol.
6. Dip 1-2x in eosin.
7. Dip 5-10x in 95% alcohol, then repeat using 2nd aliquot.
8. Dip 5-10x in 100% alcohol, then repeat using 2nd aliquot.
9. Dip 5-10x in xylene, then repeat using 2nd aliquot.
10. Apply coverslip and seal.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Sequence Read Archive (SRA) is a database for biological sequence data and is maintained by the National Center for Biotechnology Information (NCBI). Sequence files can be obtained by using the SRA Toolkit. This protocol provides the steps necessary to use the SRA toolkit to get sequence data in fastq format. </p>

[BEFORE_START]
<p>Login to the UA hpc. This protocol will begin in your home directory. </p>

[GUIDELINES]
<p><a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc" target="_blank">SRA Toolkit Documentation</a></p>

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

[BEFORE_START]
Thaw GeltrexTM Basement Membrane Matrix at 2-8°C overnight. Refrigerator temperatures may vary; therefore, thaw extract on ice in a refrigerator. Basement Membrane Matrix gels in 5-10 minutes above 15°C; therefore when working from a full 5 ml vial, it is unnecessary to keep it on ice if used within 5 minutes and the environmental temperature does not exceed 25°C. It is also unnecessary to prechill pipette tips, tubes, plates, or other objects that may come in contact with the extract. Since smaller volumes warm more quickly, partial tubes and aliquots should be kept on ice to prevent premature gelling. 

[GUIDELINES]
Geltrex Catalog NO: A1413302 (5 ml from ThermoFischerScientific). Thin Gel Method (non-gelling) for hESC Applications. From: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157883/ 
 
 
 
 
 

[STEPS]
SECTION: Thawing of Geltrex, dilution in DMEM/F12 and aliquotting for storage at -20C
?. 
SECTION: Plate coating
?.
TITLE: Intraperitoneal Injection in an Adult Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the general procedure used for intraperitoneal injection in adult mice.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: FACS Single Cell Sorting
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides step by step instructions for performing single-cell sorting by using BD FACSAria II machine or BD FACSAria Fusion machine and BD FACSDiva software.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Automated high throughput viral concentration from wastewater using the KingFisher Flex platform
AUTHORS: Smruthi  Karthikeyan, Greg Humphrey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Large-scale wastewater surveillance has the ability to greatly augment the tracking of infection dynamics especially in communities where the prevalence rates far exceed the testing capacity. However, current methods for viral detection in wastewater are severely lacking in terms of scaling up for high throughput. In the present study, we employed an automated magnetic-bead based concentration approach for viral detection in sewage that can effectively be scaled up for processing 24 samples in a single 40-minute run. The method compared favorably to conventionally used methods for viral wastewater concentrations with a limit of detection of 8.809 viral gene copies/ml from input sample volumes as low as 10ml and can enable the processing of over 100 wastewater samples in a day.</div></div>

[STEPS]
?. Aliquot  of raw sewage (no pre-filtering step required) into two wells of the KingFisher 24 deep-well plate (Cat# 95040470, Thermo Fisher). Each well should have  of the raw sewage sample. For instance,    of sample PL09 should be filled in A1 of plate 1 and   in A1 of plate 2.
10 mL
5 mL
5 mL
5 mL
?. Add  of   to each of the wells containing the raw sewage sample.
75 µl
?. Prepare the elution plate: Add  of 1X PBS and  of the to each of the wells of the 24 deep-well plate.
150 µl
800 µl
?. Load the 3 plates into a  KingFisher™ Flex Automated Nucleic Acid Purification system fitted with a 24 Deep-Well Head for KingFisher™ Flex Magnetic Particle Purification System (Cat. # 5400630 and 5400640).
?. Download the program for the KingFisher Platform for the automated processing of 24 samples:  and load the program on the the equipment using the BindIt Software (Cat. # 5189009).
?. Once loaded, follow the steps on the the protocol to load the 3 plates into the system for extraction.
The protocol provided here includes an extended incubation step which can be reduced by half depending on the sample volume. The above protocol can also be increased for processing 48 samples.
?. The program processes the samples from the 2 sample plates and elutes the concentrated viral RNA into a single elution plate containing the lysis buffer.
The Lysis buffer can vary depending on the Nucleic extraction kit used.
?. Nucleic acid extraction can then be performed using the
The downstream extraction can be performed with any kit of choice. Here the MagMAX kit was used  due to ease of integration into the KingFisher Flex system.
TITLE: Step 1: Swab sample collection
AUTHORS: Peter H L Krijger, Tim A Hoek, Sanne Boersma, Lieke I P M Donders, Maaike M C Broeders, Mark Pieterse, Pim W Toonen, Ive Logister, Bram M P Verhagen, Marjon J A M Verstegen, Thomas W van Ravesteyn, Rene J T M Roymans, Francesca Mattiroli, Jo Vandesompele, Monique Nijhuis, Stefan Meijer, Anton van Weert, Edwin Dekker, Fred J Dom, Rob Ruijtenbeek, Lieven B J van der Velden, Jeroen H B van de Bovenkamp, Martijn Bosch, Wouter de Laat, Marvin E Tanenbaum

[DESCRIPTION]
One of the most critical innovations within STRIP is the collection of samples in small tubes (0.8 ml compared with 5–15 ml in standard diagnostics) with virus-inactivating and RNA-stabilizing lysis buffer (InActiv Blue) spiked with a control RNA (PDV). The use of small tubes enables the collection of sample tubes in small 96-tube boxes at sample collection sites. These 96-tube boxes are directly compatible with automated sample processing in the lab without slow and often laborious transfer of samples from collection tubes to plates that are compatible with robotics.






[BEFORE_START]
Collection personnel should be trained in the proper collection procedure.

[STEPS]
SECTION: Sample collection
1. Person 1 scans the 2D barcode at the bottom of a 0.8 ml Micronic tube prefilled with 0.4 ml virus transport buffer ( InActiv Blue mixed with a spike-in control RNA) and places it in a Micronic tube rack (tube rack A).
SECTION: Sample collection
2. Person 1 removes cap from the Micronic tube using the manual recapping device (cap stays on the recapping device).
SECTION: Sample collection
3. After instructing the patient to blow their nose, the naso- oropharyngeal swab specimen is collected by person 2 using 1 flocked swab using standard procedures. Importantly: Make sure no mucus is attached. Repeat swab collection when needed.
SECTION: Sample collection
4. Person 2 places the swap in the tube placed in tube rack A, rotates the swab 10 times between thumb and index finger, and trashes the swab.
SECTION: Sample collection
5. Person 1 close the tube with the cap and transfers the tube to a new tube collection rack (tube rack B).
SECTION: Sample collection
6. Store at room temperature until shipment to the RNA extraction site. Samples are processed as soon as possible after arrival at the lab. Samples are regularly processed within 24h but can be stored up to 72h if required.
TITLE: Investigating DOIs&#39; classes of errors
AUTHORS: Ricarda Boente, Deniz Tural, Cristian Santini, Arcangelo Massari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to describe an automated process to repair invalid DOIs. In particular, four classes of errors are addressed: previously invalid DOIs become valid, prefix errors, suffix errors, and other type errors.</div></div>

[STEPS]
?. [Checking  the DOI names' invalidity]
We import a CSV containing invalid DOI-to-DOI citations organized as follow: the first column contains valid citing DOIs and the second contains invalid cited DOIs
?. [Checking  the DOI names' invalidity]
First, we check for each cited DOI if it is factually invalid. For this purpose, the DOI Proxy is used (https://www.doi.org/factsheets/DOIProxy.html): if the status code corresponding to that specific DOI is different from 1, it means that it is not valid; otherwise, that DOI has become valid in the meanwhile and the algorithm returns the same DOI as a correct one.
?. [Error analysis]
Taking as a reference the DOI error's taxonomy by Buchanan (Buchanan, 2006), there are two main classes of errors: author errors, made by authors when creating the list of cited articles for their publication, and database mapping errors, related to a data-entry error. This protocol deals only with the second kind of error, which can be further divided between prefix errors, suffix errors, and other-type errors (Xu et al., 2019). In order to solve our problem, we isolated recurrent strings at the beginning and at the end with corrupted DOI prefix and suffix respectively. In addition, we found other types of errors, like wrongly encoded characters and unwanted characters, that could be removed at the end of the data cleaning process.
?. [Data cleaning process]
Here we describe the steps through which we carried out the cleaning process of factually invalid DOIs
?. [Data cleaning process]
We define two regular expressions: one for identifying corruptions at the beginning of the DOI and one for cleaning corruptions at the end.
?. [Data cleaning process]
For each factually invalid DOI, we apply the two regular expressions for cleaning prefix-type and suffix-type errors
?. [Data cleaning process]
Once we have cleaned the DOI from the aforementioned error types we remove unwanted characters, that is double underscores, double periods, XML tags, spaces, and forward slashes.
?. [Data cleaning process]
After this procedure, we store the modified DOIs in an output CSV file, where for each cited DOI we also store a value corresponding to the error class to which it belongs.
?. [Checking the modified DOI names]
Finally, each modified DOI name is checked through the DOI Proxy, to verify if the aforementioned procedure was able to fix it or not. In the end, we provide the number of DOI names that we were able to fix and the number of DOI names for each class of errors.
TITLE: Yeast transformation
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for the transformation of yeast cells with linear or circular DNA.</div></div>

[STEPS]
?. Make a transformation mix consisting of:  AB1DNA34 µL (0.5-4 µg per fragment)2Salmon sperm DNA 2 mg/mL50 µL3PEG-3350240 µL41M LiOAc36 µL5MQ waterup to a final volume of 360 µL
AB1DNA34 µL (0.5-4 µg per fragment)2Salmon sperm DNA 2 mg/mL50 µL3PEG-3350240 µL41M LiOAc36 µL5MQ waterup to a final volume of 360 µL
?. Centrifuge the 1.5 mL tubes with 100 µL of yeast competent cells for 2 minutes at 5000x g and remove the supernatant.
?. Add 360 µL of the transformation mix into the 1.5 mL with the cells and vortex trhoughly.
?. Heat-shock the solution at 42 ºC for 40 minutes.
?. Centrifige the cells for 2 minutes at 5000x g and remove the supernatant.
?. Resuspend the cells in 1 mL of YPD medium. Divide the volume into two different 1.5 mL tubes.
?. Leave cells to recover for 1.5 hours at 30 ºC and 300 rpm.
?. Centrifuge cells for 2 minutes at 5000x g and decant supernatant, leaving a bit of the medium inside the tubes.
?. Resuspend cells in the resting medium and plate 50 µL onto a SC plate without the amino acid used as a selection marker.
?. Incubate the cells for 3 days at 30 ºC.
TITLE: Gene editing of YIPF4 in hESCs V3
AUTHORS: Harper JW, Kelsey Hickey, sharan_swarup

[DESCRIPTION]
This protocol describes the creation of YIPF4 knockout hESCs.

[STEPS]
SECTION: Cell line maintenance
1. Maintain H9 human embryonic stem cells (AAVS1-TRE3G-NGN2 hESCs) in complete (E8 media).
SECTION: Targeted knock-out of YIPF4
2. For YIPF4 knock-out, the following sgRNA sequences was designed and ordered from Synthego (5’  AAGAGGTTATGGCTGGCTTC 3').

SECTION: Targeted knock-out of YIPF4
3. 0.6 μg sgRNA was incubated with 3 μg SpCas9 protein for 10 mins at room temperature and electroporated into 2 × 105 H9 cells using Neon transfection system (Thermo Fisher Scientific). 


SECTION: Targeted knock-out of YIPF4
4. Deletions were verified by DNA sequencing with Illumina MiSeq (F: GTTCAATAGCATCCCCAGATGT and R: TCATCCCACAGACTAGCTCAAA) and by immunoblotting with a YIPF4 antibody (Sino Biological 202844-T46).
TITLE: Serial Dilution of Nucleofected iPSC Pools
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Coat 3 wells of a 6 well plate with  Matrigel (supplemented with RGD fragment)
1 ml
?. Aspirate media from cells in culture.
?. Wash with - PBS per well.
1 ml
2 ml
?. Add  Accutase per well.
1 ml
?. Incubate at for  to achieve single cells.
Individual donor lines exhibit variable sensitivity to accutase-mediated dissociation. Monitor cells regularly to identify when cells achieve single cells.
?. Collect cells in 5 mL DMEM/F12 and transfer to a 15mL conical tube.
?. Spin at 750-800 rpm for .
?. Aspirate media.
?. Resuspend cells in mTesR1 supplemented with 5 uM Rock Inhibitor.
?. Plate several dilutions of cells over the three wells (typically 25 μl, 50 μl and 75 μl in 2 mL of mTesR1).
?. Freeze down the remaining cells by adding an equal volume of 2x iPSC Freezing Media (20% DMSO in FBS).
?. Incubate at  overnight.
?. Change mTesR1 the following day.
TITLE: HiFi Gibson Assembly (Protocol for the NEBuilder® HiFi DNA Assembly Master Mix)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol for DNA Assembly using the NEBuilder® HiFi DNA Assembly Master Mix.</div></div>

[STEPS]
?. Set up the following reaction on ice (to 20μl total volume):  ABC12-3 Fragment Assembly4-6 Fragment Assembly2DNA RatioVector:Insert = 1:2Vector:Insert = 1:13Total amount of Fragments0.03-0.2 pmols0.2-0.5 pmols4NEBuilder HiFi DNA Assembly Master Mix10 μL10 μL5Deionized H2O10 - X μL10 - X μL6Total Volume20 μL20 μL
ABC12-3 Fragment Assembly4-6 Fragment Assembly2DNA RatioVector:Insert = 1:2Vector:Insert = 1:13Total amount of Fragments0.03-0.2 pmols0.2-0.5 pmols4NEBuilder HiFi DNA Assembly Master Mix10 μL10 μL5Deionized H2O10 - X μL10 - X μL6Total Volume20 μL20 μL
?. Incubate samples in a thermocycler at 50ºC for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled).
?. Following incubation, store samples on ice or at –20ºC for subsequent transformation.
?. Transform into chemically competent cells (1-5µL) or in electrocompetent cells (1 µL, diluted 10x or 5 µL, after purification to remove salts).
TITLE: 10x Protocols: Visium v2 CytAssist FFPE Library Construction -- University of Minnesota TMCs (CG000495 Rev D)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v2 chemistry on FPPE samples with the CytAssist component.

Protocol ID# (CG) and Revision used: 
    10x protocol CG000495, Revision D -- Library Construction with CytAssist

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. 10x protocol CG000 495, Revision D (Library construction with CytAssist)
  

2. Additional Protocols/Resources
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe