eamag/protocols-with-steps huggingface dataset:
TITLE: iPSC gDNA Extraction: For Screening Edited Clones
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Spin 96 well PCR plate at 3800 rpm for  at .
?. Pipet off supernatant.
If worried about removing cells, transfer supernatant with multichannel pipet into new PCR plates and store at   until DNA extraction is complete.
?. To cell pellet (often not visible), add  QuickExtract DNA Solution (Epicentre Technologies QE09050).
50 µl
?. Vortex plate for.
?. Incubate plate at  for .
?. Vortex plate for .
?. Incubate plate in thermocycler at  for .
?. Place plate in  for storage, until ready to use for further screening experiments .
TITLE: Filter trap assay for the detection of alpha-synuclein aggregation
AUTHORS: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl

[DESCRIPTION]
This protocol describes an assay that detects aggregated alpha-synuclein in cell lysates.


[STEPS]
SECTION: Harvesting cells
1. Prepare lysis buffer and place on ice.

SECTION: Harvesting cells
2. After 1440 min of exposure to PFFs/PBS, wash wells 1X with 1 mL warm PBS.

SECTION: Harvesting cells
3. Withdraw PBS and treat wells with 300 µL of TrypLE.

SECTION: Harvesting cells
4. Quench dissociation reagent with 300 µL of 10% FBS and transfer the contents of the wells to 1.5 ml tubes.

SECTION: Harvesting cells
5. Centrifuge 1500 x g for 3 min.

SECTION: Harvesting cells
6. Withdraw the supernatant and wash cells with 1 mL PBS.

SECTION: Harvesting cells
7. Centrifuge 1500 x g for 3 min.

SECTION: Harvesting cells
8. Withdraw supernatant and resuspend in 100 µL of lysis buffer and incubate on ice for 5 min.

SECTION: Harvesting cells
9. Sonicate the cells in the Bioruptor for 3 cycles of 30 s on and 30 s off at high power.

SECTION: Harvesting cells
10. Centrifuge the lysate at 500 x g for 5 min and place the clarified lysate in a new 1.5 ml tube.

 

 

SECTION: Harvesting cells
11. Determine the concentration of protein in each sample by Pierce Rapid Gold BCA Protein Assay.
SECTION: Filter Trap
12. Soak the cellulose acetate membrane in wash buffer for > 10 min.

SECTION: Filter Trap
13. For each well that you run, prepare 1020 µL of the sample containing 10.2 - 51 ug of lysate, with the remaining volume consisting of lysis buffer.

SECTION: Filter Trap
14. Assemble the slot blot vacuum manifold apparatus with the cellulose acetate membrane.
SECTION: Filter Trap
15. Wash each well with 300 µL wash buffer, inspecting each well to ensure that the buffer can drain through it.

 

SECTION: Filter Trap
16. Load 1000 µL (10 - 50 ug) of sample to each well.

SECTION: Filter Trap
17. Once the sample has drained through the wells, wash each well 3 times with 300 µL of wash buffer.

SECTION: Filter Trap
18. When all of the washes have drained through the wells, trim the membrane and proceed with standard immunodetection methods.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol outlines the analysis used to plot KEGG functional annotation.</p>

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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TITLE: Real-Time PCR protocol to screen for SARS-COV-2 variants of concern (B.1.1.7, P.1 and B.1.1.35) 
AUTHORS: Camila Romano, Jaqueline De Jesus, Alvina Clara Felix, Anderson V de Paula, Pâmela S Andrade, Franciane M de Oliveira, Darlan Cândido, Nuno Faria, William M  de Souza, Ester C Sabino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The recently emerged variants of concern (VOC) B.1.1.7 in UK, B.1.1.35 in South Africa and P.1 in Brazil have increased transmissibility and are rapidly spreading worldwide. These variants have several substitutions that allow them to bypass the immune responses and are up to 10 times more contagious than the previous circulating SARS-COV-2 lineages. While in some regions P.1 predominates (as several estates in Brazil), in other countries B.1.1.7 or B.1.1.35 are most prevalent.  Therefore, real-time surveillance of VOC is the key for rapid public health responses. In this matter, RT-PCR protocols that are capable of detecting VOCs easily and rapidly are needed. We developed and validated a protocol that detect simultaneously the 3 most widespread VOC. Although this protocol does not discriminate among the VOCs, it can reliably detect mixed infection. Importantly, our protocol successfully identified a co-infection by two SARS-COV-2 lineages (P.1 and B.1), confirmed through complete genome sequencing.</div></div>

[STEPS]
?. Reconstitute the oligos/probes to 100uM with TE or nuclease-free water. Vortex briefly and allow to sit for about 2 minutes.
?. Prepare 10uM working solutions (primers and probes). Suggestion: if the original concentration is 100uM, add 10ul of the reagents in 90ul of ultrapure nuclease-free water.
?. The reaction was not adjusted for multiplex use, so prepare the reactions separately. We recommend running the probes NSP6 Wild and NSP6 Delete in the same plate/experiment for better results.
?. Prepare your Master mix as the following (use primers and probes at 10uM)ReagentsVolume 1 reaction (ul)x1004x TaqMan Fast Virus 1-Step Master Mix5500NSP6_Forw (300nM/reaction)0.550NSP6_Rev (300nM/reaction)0.550Probe (300nM/reaction)0.550RNA8.5-Total15-
ReagentsVolume 1 reaction (ul)x1004x TaqMan Fast Virus 1-Step Master Mix5500NSP6_Forw (300nM/reaction)0.550NSP6_Rev (300nM/reaction)0.550Probe (300nM/reaction)0.550RNA8.5-Total15-
?. Add 6.5ul of the mix to each well and 8.5ul of the RNA template. Don't forget using positive and negative controls.
?. In case of bubbles, spin the plate before cycling.
?. Run the following cycling at the Real time machine:50°C2 min95°C10 min95°C15 sec40x60°C1 min
50°C2 min95°C10 min95°C15 sec40x60°C1 min
?. Interpreting results: NSP6_WildNSP6_DeleteResultCT ≤ 38CT > 39/undetectednon- VOCCT > 39 /undetectedCT ≤ 38VOC (B.1.17; P.1 or B.1.1.35)CT ≤ 38CT ≤ 38mixed infectionCT > 39/undetectedCT > 39/undetectedinvalid/negative
NSP6_WildNSP6_DeleteResultCT ≤ 38CT > 39/undetectednon- VOCCT > 39 /undetectedCT ≤ 38VOC (B.1.17; P.1 or B.1.1.35)CT ≤ 38CT ≤ 38mixed infectionCT > 39/undetectedCT > 39/undetectedinvalid/negative
TITLE: Autoclave Balch Tube Sterilization
AUTHORS: Ada de la Cruz, Catherine Gohar

[STEPS]
?. [BACTERIAL]
Place tubes that need to be sterilized into an autoclave tub.
?. [BACTERIAL]
Fill tub with tap water and add soap solution to help labels come off.
?. [BACTERIAL]
Place autoclave tub with tubes into autoclave and select the "P12 30 min liquid" program.
?. [DECRIMPING]
Once out of autoclave, place the tubes into a bin and decrimp the stoppers. Pour the media down the drain with plenty of running water.
?. [DECRIMPING]
Place the empty tubes and stoppers in the sink and scrub with special detergent + tap water. Ensure the inside has no remaining cell debris or medium residue.
?. [DECRIMPING]
Rinse the tubes and stoppers with RO water.
?. [DISHWASH]
Place the stoppers in the dishwasher. Select wash cylce #4 and start.
?. [DISHWASH]
Soak the tubes in 10 mM HCl sol'n for one day. (If there is not enough room for all the tubes in the HCl container, keep remaining tubes soaking in RO water + label "pre-HCL RO").
?. [DISHWASH]
Rinse tubes with RO water 3x.
?. [DISHWASH]
Put tubes in dishwasher using the plastic tube dishwasher cartons. Select wash cycle #4 and press start.
?. [NANOPURE]
Remove tubes from dishwasher, rinse 3x in nanopure water, then soak them for at least two hours (label post-wash nanopure); one day is optimal.
?. [NANOPURE]
If you haven't done so, remove stoppers from dishwasher and rinse with nanopure water.
?. [NANOPURE]
Place everything in plastic dishwashing cartons and let air dry.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This collection of files contains the raw data and R script for the paper entitled 'The UK Research Excellence Framework and the Matthew Effect: Insights from machine learning.'
 
 
Data:
The file balbuena_REF_2014.dta is a Stata 12 file that contains the data for the paper. It is also saved in CSV format as Balbuena_REF_2014.csv
The file balbuena_ref_syntax.R contains the syntax used to run the analysis.
The file balbuena_REF.RData file contains the data objects in RData format. See the instructions below if you wish to replicate the analysis.  To simply view the data in Excel without re-running the analysis, the universities in the training set (n  = 79) and testing set (n=30)  are provided in the excel file “REF_2014_schools.xls”
 
 

[STEPS]
?. 
?. 
?. 
SECTION: Open the syntax file in R / Rstudio
?.
TITLE: Study Protocol of PISQ-12 Indonesian Version  
AUTHORS: Suhair Quzwain, Tyas Priyatini, Aulia Khatib

[DESCRIPTION]
1. Translation Process. We obtained PISQ-12 questionnaire from the English version of Int Urogynecol J Pelvic Floor Disfunct. 2004 May-Jun;15(3):219. We sent an email to Rebecca G. Rogers, MD asking for permission to translate and test the validity and reliability of the questionnaire into Indonesian language. The questionnaire went through forward – backward translation procedure. In this procedure, the translation process was converted from English into Indonesian language. The translation results were translated back from Indonesian into English by different translator.
2. A meeting was conducted to make concise judgement and review for the questionnaire using the face validation method and discussions between 2 translators and 6 urogynaecologists (reconstructive urogynaecology consultants) regarding the results of the translation for each question in this questionnaire.
3. We conducted an initial test (pilot study) towards 10 sexually active female subjects who had agreed to participate in the study by filling out the Indonesian version of the PISQ-12 questionnaire to see understanding, filling in time and difficulties experienced in answering questions on the questionnaire. A supervisor was assigned if participants faced any problems throughout this process.
4. Conduction of primary research. Respondents were recruited from patients diagnosed with pelvic organ prolapse and/or urinary incontinence at the RSCM urogynecology polyclinic who agreed to participate in the study (signed written informed consent). Subjects who met the criteria and were willing to take part in the study were then collected through medical records and filled out the Indonesian version of the PISQ-12 questionnaire through guided interviews by researchers or research assistants. Filling out of the second questionnaire was carried out 2 weeks later.
5. Data analysis. Data analysis was carried out to test the statistical validity and reliability of the questionnaire. We used Pearson test to get the value of r. If the r test value is greater than the r table value, the result was declared valid. The reliability test was carried out by looking for the alpha value with the Cronbach's alpha test. If the alpha value was greater in the r table, then the questionnaire was declared reliable.

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

[DESCRIPTION]
This is a protocol that describes the process of isolating single cells from human knee meniscus for scRNA-seq.

[STEPS]
1. ~1g of tissue from healthy donor knees (Grades 0-1) is collected from the meniscus. For details regarding the tissue harvesting procedure please see 
dx.doi.org/10.17504/protocols.io.6qpvr614zvmk/v1 
2. Meniscal tissue is washed with Room temperature   supplemented with 10%  , 1%   and 1%  .

3. The tissue is then finely minced with a #21 Feather disposable scalpel, and digested in 20mL   supplemented with 1% Anti-Anti and 2%   using 100 rpm shaking at 37 °C for 30 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

10. 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  .

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

12. The supernatant is carefully removed, and the remaining cell pellet is delicately resuspended in 10 mL of DPBS supplemented with 0.04%  .

13. 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.
6. Carefully remove and collect the collagenase supernatant; the collagenase is reused in subsequent steps.  
7. Cells are then resuspended in DMEM supplemented with 10% CS, 1% Anti-Anti and 1% PSG and stored at 37 °C. 

8. Repeat the digestion, spin down and filtration steps for 60 min and then 120 min, for a total of 210 min of digestion.

9.  Combine all collected cells and spin down at 1200 rpm for 5 min at Room temperature.
TITLE: GDSC data set and random forest model scripts 
AUTHORS: Peter Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This location contains the GDSC data set with 145 oncogene mutation statuses and ~1200 chemical descriptors. For instructions how to run binary classification (to predict compound activity vs inactivity) or regression (to predict values of log IC50), see the README.classification.txt and README.regression.txt files, respectively.</div></div>

[STEPS]
TITLE: Fascial manipulation® for musculoskeletal disorders: a scoping review
AUTHORS: Yuichi Isaji, Daisuke Sasaki, Yusuke Kon, Takashi Kitagawa

[DESCRIPTION]
Objective: The purpose of this scoping review is to synthesize all available literature on the effects/mechanisms of myofascial manipulation interventions and adverse events, to assess knowledge gaps in understanding optimal approaches, and to elucidate therapeutic mechanisms and select indications.    

Introduction: Fascial manipulation® (FM) is one of the techniques that have been developed in recent years as a manual therapy for musculoskeletal disorders. Systematic reviews have shown the efficacy of FM for patients with musculoskeletal pain, but mechanisms and adverse events have not been examined.

Inclusion / Exclusion criteria: This review will include patients with musculoskeletal disorders with pain and dysfunction. Outcomes will be pain and physical function. The study design will be that of an interventional trial or observational study or case report, with no restrictions on region, race, gender, or language of the original paper.

Methods: A systematic search of PubMed, CINAHL, Web of Science, and PEDro databases using the keywords “Fascial manipulation®,” “musculoskeletal disorders,” and “myofascial pain” and “musculoskeletal pain” during December 2022. In the first screening step, two independent reviewers will review all the titles and abstracts to exclude irrelevant articles. In the second screening step, two independent reviewers will review all the full texts to exclude irrelevant articles. Outcomes will be focused on pain and physical function. Chapters will be divided for each outcome and the results will be described.

[STEPS]
1.
TITLE: User Protocol for SaliCoV Detection Kit
AUTHORS: huan.jia , patthara 

[STEPS]
?. Turn on 'Ninja Eyes' device, put a white paper on cassette loading tray. Wait for device to warm up till user interface is lighted up.
?. Transfer test individual's saliva sample from saliva collection tube to Reagent A bottle with provided Small, Clear 100 uL Fixed Volume Pipette.
[Reagent A is stored at 4°C before usage.]
100 µl
?. Recap Reagent A bottle with dispensing cap and wait for 1 minute.
?. Apply sample in Reagent A bottle to 'T' and 'C' wells on test cassette. One drop for each well.
[Each drop from Reagent A bottle is 40 uL.]
?. Apply Reagent B to 'T' and 'C' wells immediately after step 4. One drop for each well. NOTE: Do not let tip of Reagent B bottle to touch wells on test cassette.
[Each drop from Reagent B bottle is 40 uL.]
?. Load test cassette into 'Ninja Eyes' device loading tray immediately after step 5.Press 'START' button on device's touch screen.
?. 'Ninja Eyes' device will beep after 3 minutes when result is ready. Discard test cassette as biohazard waste.
TITLE: URA3 PCR
AUTHORS: Brian    Teague

[DESCRIPTION]
 The polymerase chaine reaction (PCR) amplifies linear DNA using a DNA polymerase enzyme and a pair of short single-stranded DNA "primers." This protocol is for amplifying the URA3 gene from the YTK74 plasmid and adding the homology sequences needed for homology-directed repair.

  


[STEPS]
1. Check that the thermocycler is programmed and holding at 98°C. The thermocycler program we're using is the following:


 


 

2. Grab an ice bucket and fill it with ice. If you don't have an ice bucket, a beaker will work in a pinch.
3. If necessary, dilute the primers to a concentration of 10 micromolar (µM) in   . (Make 100 µl of each dilution.)

 

4. Mix the following in a PCR tube on ice, in this order:
7 µL   
1 µL forward primer
1 µLreverse primer
1 µL template DNA
10 µL   







5. Mix the reaction by gently flick the tube several times, then spin down in a microfuge.

 

6. Transfer the tube from ice to a pre-heated thermocycler holding at 98°C. Start the PCR program.
7. After the PCR program has run, store the tube at -20°C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 8">
<div>
<div>
<ol style="list-style-type: none;" start="21">
<li>
<p>The royal python (<em>Python regius</em>) is commonly bred in captivity. To have a successful breeding, accurate monitoring of the reproductive activity is necessary. The use of non-invasive monitoring methods in exotics is important in order to minimize stress. For this purpose, ultrasound has been anecdotally used to monitor royal python reproductive activity. However, there is limited information regarding the reproductive cycle of this species. The aim of the present study is to monitor the female reproductive cycle of the royal python using ultrasonography and gonadal steroid metabolite measurements in the faeces. The reproductive activity of one hundred twenty-nine adult female <em>P. regius</em> was examined during two consecutive years. We performed brief scans on non-anaesthetized snakes using a portable ultrasound system and a 10-12 MHz linear array transducer (MyLab™ 30 Gold, Esaote). Ultrasound features, dimension and echogenicity of the reproductive structures were determined. During the second reproductive cycle, the hormonal profiles of 30 animals were also evaluated, with a monthly collection of faecal samples. These samples were classified according to reproductive stage, and the mean faecal progesterone and 17β-estradiol levels were calculated using the results from an enzyme-linked immunosorbent assay (ELISA). Progesterone levels increased during the reproductive cycle. estradiol levels showed greater variability, although they appeared to increase before coupling when compared to the levels between coupling and egg laying. The present study suggests that it is possible to identify different phases in the female royal python reproductive cycle: anovulatory phase, transition, folliculogenesis and embryogenesis. Ultrasound is also useful for identifying follicular regression or slugs. Gonadal steroid metabolite measurements from the faeces could help integrate the reproductive information. The use of ultrasonography in addition with the steroid metabolite measurement in the faeces gives an accurate picture of ovarian activity in captive adult female royal pythons. </p>
</li>
<li></li>
</ol>
</div>
</div>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A modified TOTO-1 iodide labeling protocol to enable visualization of nucleic acid loading efficiency, as a means to verify that nucleic acid constructs are entering the cells. With additional cleanup steps to decrease background staining. A convenient assay to use in conjunction with transfection optimization. (Modified from Golzio, M., Teissie, J. &amp; Rols, M.P. 2002. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99, 1292–1297.)</p>

[GUIDELINES]
<table width="637">
<tbody>
<tr>
<td colspan="4" width="376">DNA Copy Number Calculation and Dye Binding Ratio</td>
<td width="87"> </td>
<td width="87"> </td>
<td width="87"> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">DNA Size, bp</td>
<td> enter value</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">DNA Conc, ug/uL</td>
<td> enter value</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">DNA Abundance, copies/uL</td>
<td colspan="5">=((DNAConc, ug/uL)*6.023*1E+23)/(DNA Size, bp*1000000*650)</td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>bp per vol</td>
<td colspan="4">=(DNA Abundance, copies/uL)*(DNA Size, bp)</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">Binding Target</td>
<td>5 bp/dye</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">Dye molecules needed</td>
<td colspan="3">=(bp per vol)/(Binding Target)</td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">Dye Conc, M</td>
<td> enter value</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>molecules/L</td>
<td colspan="2">=Dye Conc, M*6.023E+23</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td width="87">Dye Volume Needed</td>
<td>=(Dye molecules needed/molecules per L)*1000000 uL Dye per uL DNA</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>

[STEPS]
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