eamag/protocols-with-steps huggingface dataset:
TITLE: Qiagen RNEasy PowerMicrobiome RNA extraction kit
AUTHORS: Michael Dan Siemon, Christelle Schang et al, Jessica Pardy, Richard Gibson, Dilan Joseph, Justin Donovan, christopher.degroot

[DESCRIPTION]
The samples were processed using the Qiagen RNeasy PowerMicrobiome kit with the modifications described by Schang et al., 2021. As a substitute for vortexing described in the kit protocol, bead-beating was used. 100ul of phenol-chloroform-isoamyl alcohol was added to the bead beating tubes before the addition of the membranes. Bead-beating was conducted 4x for 30s at 4 m/s. After pelletizing the samples with centrifugation, the supernatant was processed through spin columns. Spin columns were incubated with DNase Digestion Solution for 15 minutes to isolate only RNA. The RNA was eluted from the spin columns by passing 50 uL of DEPC water through twice. 

[STEPS]
SECTION: Qiagen RNEasy PowerMicrobiome RNA
4. As a substitute for vortexing described in the kit protocol, bead-beating was used. Bead-beating was conducted 4x for 30s at 4 m/s.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
2. Add 100 µl phenol–chloroform–isoamyl alcohol to a PowerBead Bead Tube, Glass 0.1 mm. Place 0.25 g stool or biosolid sample into the Bead Tube. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
1. Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 4500 x g for 20 min. Decant supernatant, assume 280 µl pellet.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
5. After pelletizing the samples with centrifugation (13,000 x g for 1 min), mix the supernatant with 650 µl each of Solution PM3 and Solution PM4. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
3. Add 650 µl PM1 and 6.5 µl β-mercaptoethanol to the PowerBead Tube
SECTION: Qiagen RNEasy PowerMicrobiome RNA
9. Incubate spin columns with DNase Digestion Solution for 15 minutes at room temperature to isolate only RNA.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
6. Process the solution through MB RNA Spin Column by centrifugation (13,000 x g for 1 min). Discard flow-through and repeat until all the solution has been processed. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
7. Shake solution PM5, add 650 µl to the Spin Column and centrifuge (13,000 x g for 1 min).
SECTION: Qiagen RNEasy PowerMicrobiome RNA
8. Conduct a drying step by centrifuging at 13,000 x g for 1 min to remove residual wash.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
14. Elute RNA from the spin columns into 1.5 ml eppendorf tubes by passing 50 uL of DEPC water through twice. Centrifuge each elution 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
11. Discard flow-through. Add 650 µl Solution PM5. Centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
12. Discard flow-through. Add 650 µl Solution PM4. Centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
10. Add 400 µl Solution PM7 and centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
13. Conduct a drying step by centrifuging at 13,000 x g for 2 min.
TITLE: Hepatorenal index protocol
AUTHORS: fabio10stahl , Fabio Lucio Stahlschmidt

[STEPS]
?. Each patient fasted for 4 h before the examination. First, the radiologist performed a comprehensive thoracic and abdominal ultrasound examination using the same ultrasound machine for all patients (S2000 HELX; Siemens, Erlangen, Germany). For all examinations, the conditions were as follows: frequency of 4.00 MHz, dynamic range of 70, grayscale map “D”, the software Advanced SieClearTMfor spatial compounding at a level 5, and the software Dynamic TCETMfor speckle reduction at a level “HIGH.”
?. Next, a photo containing the longitudinal axis of the right kidney and the liver was obtained after automated gain adjustment in the dorsal decubitus position or in the left lateral decubitus position using the subcostal or intercostal window.
?. A single focal zone was placed near the center of the image containing both the liver and the middle third of the kidney.
?. The workstation for all analysis using Osirix MD version 1.6 (Pixmeo Sarl, Swiss) after taking a 0.10 to 0.30 cm2 region of interest (ROI) brightness measurement in the liver and renal cortex. The site of the renal cortex ROI was outside or between the renal pyramids in the center of the image, not crossing a limit of 1.0 cm from the focal zone line. The liver’s ROI also did not cross a limit of 1.0 cm from the focal zone line in the center of the image. The measurements were not taken within or near the nodules and visible vessels (Fig. 1, 2).
?. Three ROIs each were obtained for the liver and renal cortex.
?. The hepatorenal index was calculated for each sequence of ROI measurements.
?. The difference between the closest indexes must be less than 0.20 hepatorenal index units, and the arithmetic mean of these closest values was then used for further analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This collection contains protocols for the computational analysis performed in the paper "The Human Skin dsDNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and its Dynamic Associations with the Host Microbiome". Geoffrey Hannigan and Jacquelyn Meisel are graduate students in Dr. Elizabeth Grice's lab in the Department of Dermatology at the University of Pennsylvania.<br /><br />Hannigan, Geoffrey D., et al. "<a href="http://mbio.asm.org/content/6/5/e01578-15" target="_blank">The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome</a>." <em>mBio</em> 6.5 (2015): e01578-15.

[STEPS]
?.
TITLE: Luminol Calibration
AUTHORS: Michael    Burgis

[DESCRIPTION]
Calibration curve using luminol in order to standardize luminescence data

[STEPS]
SECTION: Preparation of the Buffers
1. Preparation of 100ml luminol stock solution (1 mM luminol sodium salt; 50 mM sodium carbonate; 300 mM sodium bicarbonate; 5 mM ammonium carbonate) :
dissolve the following components in ddH2O
19,94mg of Luminol sodium salt
529,94mg of sodium carbonate
2,52g of sodium bicarbonate
48mg of ammonium carbonate

Preparation of the coppersulfate solution (1,5 mM CuSO4):
dissolve 23,94mg of CuSO4 in ddH2O
SECTION: Preparation of dilution series
2. Perform a dilution series in a 384 well plate similar to the protocol provided by the iGEM foundation on Fluorescein calibration:

SECTION: Preparation of dilution series
3. Add 100µl of the CuSO4 solution in the first well
SECTION: Preparation of dilution series
4. Fill every second well in that row with 50µl of water (8 rows in total)
SECTION: Preparation of dilution series
5. Transfer 50µl of the first well into the next and carefully pipette up and down to ensure proper mixing of the solutions
SECTION: Preparation of dilution series
6. Repeat this until you reach the second last well and then dispose the last 50µl
SECTION: Perform the measurement
7. Premix the luminol buffer with hydrogen peroxide in a ratio of 4:1
SECTION: Perform the measurement
8. Transfer 10µl of this solution into empty wells on the plate
SECTION: Perform the measurement
9. Use a multichannel pipette to add the inducing copper sulfate solution to the luminol solution
SECTION: Perform the measurement
10. Transfer the well plate into the plate reader and shake the plate for 30 seconds until you start the measurement
SECTION: Perform the measurement
11. Measure the luminescence with an exposure time of 250ms
SECTION: Perform the measurement
12. Plot the data in a linear correlation and use this calibration curve in order to normalize your luminescence data
TITLE: Open Field
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Open Field behavioural test.

[STEPS]
1. Habituate mice in a separate room for 60 minutes prior to testing.
2. Confirm testing room lighting is set to 100 lux and EthoVision is set up properly.
3. Place mice into open field box and record their movement for 10 minutes.
4. When testing is complete, place mice back into their home cage and clean the open field boxes well before testing the next mice.
TITLE: Mouse Genotyping with KAPA Kit in 2 Hours (#KK7302)
AUTHORS: Cq Wu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol uses KAPA Mouse Genotyping kit (#KK7302)</div></div>

[STEPS]
?. [DNA Extraction]
Collect mouse tissues (ear or tail clips) into PCR strips.
?. [DNA Extraction]
Make  extration buffer mix for each sample.  AB110x Extraction buffer10 ul2H2O88 ul3Extraction Enzyme2 ul4Total100 ul
100 µl
AB110x Extraction buffer10 ul2H2O88 ul3Extraction Enzyme2 ul4Total100 ul
?. [DNA Extraction]
Add  buffer into each tissue sample and run the reaction as follows:
100 µl
75 °C
95 °C
4 °C
Tissues are visible or intact after extraction (this is normal).Vortex DNA extract and spin down before PCR.
?. [PCR]
Make  PCR master mix for each sample.  AB12x PCR buffer10 ul2H2O8 ul310 uM Primers mix1 ul4DNA1 ul5Total20 ul
20 µl
AB12x PCR buffer10 ul2H2O8 ul310 uM Primers mix1 ul4DNA1 ul5Total20 ul
?. [PCR]
Run the reaction as follows:35 cycles35 cycles
95 °C
95 °C
[adjustable]
72 °C
[per kb]
4 °C
?. [Run DNA Gel]
Take  PCR reaction and run DNA gel.
10 µl
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocols describes materials needed and steps for maintaining coral larvae in the lab.</p>

[BEFORE_START]
<p>Materials needed:</p>
<p>Glass beakers</p>
<p>Glass bowls</p>
<p>Large glass bowl</p>
<p>Magnifying light</p>
<p>Counter for making larval counts</p>
<p>LED full spectrum lights</p>
<p>Light meter</p>
<p>HCl</p>
<p>Deionized water</p>
<p>Filtered seawater</p>
<p>Transfer pipets</p>
<p>Larval filters</p>
<p>Squirt bottles</p>
<p>Thermometer</p>
<p>Refractometer</p>
<p>Glass slides</p>
<p>Seasoned tiles (settlement substrate seasoned in field 3 months +)</p>
<p>Data spreadsheet</p>
<p>50 ml syringe</p>

[GUIDELINES]
<ol>
<li>In the morning, check lights, temperature, and salinity. Record on spreadsheet along with date and time.</li>
<li>Check traps in field in the early morning (&lt;7-8am).</li>
<li>ALL GLASSWARE MUST BE rinsed with HCl solution, DI water, and finally filtered seawater BEFORE USE! RINSE, RINSE, RINSE!!</li>
<li>Change water for all larvae daily - suck out individual larvae with pipet and place in filter (in beaker filled with filtered seawater – keep larvae underwater at all times until ready to rinse). Rinse with filtered seawater squirt bottle in filter then add to <strong>clean</strong> flask or beaker (rinsed with 10% HCl solution, DI water, and finally filtered seawater) filled with filtered seawater. Check around the edge of the filter for any trapped larvae. If larvae are not moving, very round and don’t respond after squirting or poking, they are likely dead and should be removed BEFORE adding to the new flask. Note dead larvae removed on spreadsheet.</li>
<li>Change water for all settling larvae – carefully remove half of water (be careful not to expose any slides or tiles to the air and not if any larvae have settled on the side of the beaker) and VERY carefully fill the beaker with fresh filtered sweater. Remove any dead larvae and note on spreadsheet.</li>
<li>For new larvae from field upon return, note trap on spreadsheet, empty trap contents into a large bowl and suck out individual larvae with a transfer pipet and place in filter (in beaker with filtered seawater). Rinse with filtered seawater squirt bottle in filter then add to clean flask or beaker filled with filtered seawater. Note total count in spreadsheet along with the name of person counting.</li>
<li>When finished with larvae, filter fresh seawater and clean/rinse all glassware and filters.</li>
<li>At end of day, note temperature and salinity. Record on spreadsheet along with date and time.</li>
</ol>
<ol start="9">
<li>Enter sample info into excel spreadsheet.</li>
</ol>

[STEPS]
?.
TITLE: Detection of SARS-Cov2 Without High Demand Reagents (Singleplex Assays)
AUTHORS: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In the United States, access to testing for the novel coronavirus (SARS-Cov2) is severely limited. Arguably, the PCR based tests are the most reliable when it comes to detecting the virus. Critical reagents for these tests, however, are in short supply. Our group has worked to identify and test alternative reagents and supplies that are not in high demand by clinical labs. We have adapted a more traditional approach to isolating RNA does not use a kit. RNA isolated can be used in traditional quantitative PCR or droplet digital PCR, which has been shown to be ~500 times more sensitive. </div></div>

[STEPS]
TITLE: Electroporation of Thalassiosira pseudonana
AUTHORS: Joshua Bugge, Deborah Robertson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>An electroporation-mediated genetic transformation or the marine diatom </span><span style = "font-style:italic;">Thalassiosira pseudonana</span><span> was developed. Using a sorbitol-based buffer,</span><span style = "font-style:italic;"> T. pseudonana</span><span> cells were successfully transformed with the </span><span style = "font-style:italic;">Tp</span><span>fcp/nat plasmid at an efficiency of 2820 per 10</span><span style = "vertical-align:super;">8</span><span> cells.  This  represents a six-fold improvement compared with previously published methods. </span></div></div>

[STEPS]
?. Grow cultures of T. psuedonana on f/2 supplemented sterile seawater to a density of approximately 1.1 x 106 cells mL-1
?. [Cell collection option 1]
All procedures are carried out at 4 C and cells were kept on ice. Collect cells from 500 mL of culture (see step 1)  by centrifugation for 10 m in at 3000 x g in 10 x 50 mL conical centrifuge tubes.  Discard supernatant and resuspend pellet in 1 mL of 375 mM sorbitol (filter sterilized)
?. [Cell collection option 1]
Combine resuspended cells into one 50 mL conical centrifuge tube and centrifuge at 3000 x g for 10 min.  Discard supernatant and resuspend pellet in 800 uL of 375 mM sorbitol (filter sterilized).  Transfer to 1.5 mL tube and store on ice.
?. [Cell collection option 2]
All procedures are carried out at 4 C and cells were kept on ice.Concentrate cells by vacuum filtration to approximately 300 mL.  Collect cells by centrifugation in 50 mL conical centrifugation tubes at 3000 x g for 10 min.  Discard supernatnat and resuspend pellet in in 2 mL of 375 mM sorbitol (filter sterilized).
?. [Cell collection option 2]
Combine cells into new tube and centrifuge at 3000 x g for 10 min.  Discard supernatant and resuspend in 1 mM of 375 mM sorbitol.
?. [Electroporation]
On ice, add linearized plasmid (in 250 mM sorbitol) to a final concentration of 0.15 µg mL-1 tothe  concentrated cells from above.
?. [Electroporation]
Transfer 100 µL of cells + plasmid to a pre-chilled 2 mm gap electroporation cuvette.
?. [Electroporation]
Wipe cuvette dry with kimwipe and place into Biorad Gene Pulser electroporator set to 25 µF and 400 ohm resistance. In our hands, the highest transformation efficiencies were obtained using a field strength o 2.50 kv/cm.
?. [Electroporation]
Transfer electroporated cells to 10 mL of sterile,  f/2 supplemented artiicial seawater and incubate overnight at 17 C under constant light (170 µE m-2 s-1).
?. [Electroporation]
The following day, plate ca. 5 x 108 cells on selective NEPC agar plates (in our experiment we used 100 µg mL-1 NAT).  Controls should include no-plasmid and no electroporation treatments.
?. [Electroporation]
Grow plates under constant light (170 µE m-2 s-1).  Colonies appear approximately 10 days after plating.
?. [Electroporation]
Transfer colonies to 300 µL of f/2 supplemented artificial seawater in 96 well plates. Optical denisty (600 or 680 nm) can be used as a proxy for growth using a plate reader.
TITLE: Identification of promising genotypes
AUTHORS: Ding Mingliang, Muhammad Asim, Li Mingju, Sedhom Abdelkhalik, Daniel Manore, Li Shaoxiang, Zhao Hong, Lin Liping

[STEPS]
?. Average the yield data generated for genotypes across locations and years.
?. Perform the Welch’s variance-weighted analysis of variance for studied genotypes (across years and locations), at each location (across genotypes and years) and for each year (across genotypes and locations) using One-Way ANOVA model of SAS University Edition version 3.8 with Levene test for homogeneity of variance and adopting Tukey comparison method.
?. Calculate mean genetic deviation in successive cycles
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?. Corresponding above mean values facilitate the partitioning of promising genotypes, location and years
TITLE: Promega Glucose-Glo Assay
AUTHORS: NUS iGEM

[STEPS]
?. Prepare glucose detection reagent mix by adding: Luciferin Detection Solution Reductase Reductase Substrate Glucose Dehydrogenase NAD
These reagents are part of a boxed set - Promega's Glucose-Glo Assay.
?. Dilute  overnight culture in  1x PBS and mix
2 µl
198 µl
?. Aliquot  of each sample into three wells of 96-well plate to create triplicates
50 µl
?. Add  of glucose detection reagent mix into each well
50 µl
?. Include PBS-only wells as blank
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Depending on tissue and analytes that will be analysed different matrices can be used. The following 2 matrices are first steps to analyse lipids (SDHB) and peptides (CHCA). Matrix concentration and solvent ratio as well as instrument settings can be adjusted specifically to tissue and analytes.

[STEPS]
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TITLE: Criminal Justice Policy Document Analysis . Version 2
AUTHORS: Drew Gitomer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an adaptation of Yanovitzky & Weber - because we were interested adapting Yanovitzky and Weber's tool for  criminal justice reform.


All of the policymaking process stuff was kept in, but we modified the list of policy actors, and policy focus, and Appendix C.  


We also did our coding in NVIvo so removed Appendix D in the oritinal protocol</div></div>

[STEPS]
?. [Interviews of Congressional Staff]
What information influenced your position on childhood obesity legislation?
?. [Interviews of Congressional Staff]
How did you obtain that information?
?. [Interviews of Congressional Staff]
How did you evaluate that information?
?.
TITLE: Titration of Lentivirus
AUTHORS: Anita Adami

[DESCRIPTION]
This protocol is about titrating lentivirus.

[STEPS]
SECTION: Virus transduction
1. When aliqouting the virus, prepare 1 vial with 6 μl for titration. This vial must go through one cycle of freeze / thaw before titration and if you want to titer in conjunction with aliqouting you can put the vials on dry-ice for a bit.
SECTION: Virus transduction
2. Seed cells in 6 well plates seed 100,000 cells - in 2 mL - 3 mL. For each virus  - 3x wells are required. Plate 6x additional wells for control and reference batch.

SECTION: Virus transduction
3. Dilute the virus 1:10 before adding it to the cells [54 µL + 6 µL].

SECTION: Virus transduction
4. Then add 3 μl, 10 μl and 30 μl of the diluted virus to the cells [corresponding to 0.3 μl, 1 μl and 3 μl undiluted virus].
SECTION: Virus transduction
5. Leave for 4320 min before DNA is isolated.

SECTION: Isolation of DNA
6. Look at the cells under the microscope and compare with control.
SECTION: Isolation of DNA
7. Remove media and wash 1x with 1 mL  [Use 1000 ul pipette and don´t add the PBS directly on the cells to avoid flushing them off].

SECTION: Isolation of DNA
8. Add 500 µL and leave for a few minutes in the incubator.

SECTION: Isolation of DNA
9. After incubation with trypsin, add 500 µL and transfer cells to 1.5 ml tubes.

SECTION: Isolation of DNA
10. Spin down at 300 x g, 5 min.

SECTION: Isolation of DNA
11. Remove supernatant with a pipette to not disturb the pellet.
SECTION: Isolation of DNA
12. Make a master mix of 200 µL + 20 µL per sample and resuspend the pellet in 220 µL.

SECTION: Isolation of DNA
13. Add 200 µL [w/o ethanol] and mix thoroughly by vortexing.

SECTION: Isolation of DNA
14. Bring samples to DNA isolation room and follow the instructions from the Qiagen DNeasy Protocol: Purification of Total DNA from Animal Blood or Cells [Spin-Column Protocol]. Start with incubation at 56 °C for 10 min and then move directly to step 3.

SECTION: Isolation of DNA
15. Assess titre using qPCR.
TITLE: Serial Sectioning Optical Histology - Cell Census Network -data acquisition and processing
AUTHORS: shuaibin  Chang, Jiarui Yang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">No current imaging technology can directly and without significant distortion visualize the defining microscopic features of the human brain. Ex vivo histological techniques yield exquisite planar images, but the cutting, mounting and staining they require induce slice-specific distortions, introducing cross-slice differences that prohibit true 3D analysis. Clearing techniques such as CLARITY have proven difficult to apply to large blocks of human tissue, and cause dramatic distortions as well. Thus, we have only a poor understanding of human brain structures that occur at a scale of 1-100μm, in which neurons are organized into functional cohorts. This impairs our ability to classify cell types, as the functional properties of any given cell are a function of both its molecular characteristics and the spatial context within which it resides. To date, mesoscopic features such as cortical laminae which are critical components of this spatial context, have only been quantified in studies of 2D histologic images acquired in a small number of subjects and/or over a small region of the brain, typically in the coronal orientation, implying that features that are oblique or orthogonal to the coronal plane are difficult to properly analyze. </div><div class = "text-block">Our consortium will develop and utilize an imaging infrastructure to create a human brain cell census and instantiate it in a coordinate system that will enable an immediate impact of all in vivo MRI studies of the human brain. Our imaging pipeline begins with 150 μm isotropic ex vivo structural MRIs and 500 μm 90 direction diffusion MRI. 4x4x2 cm tissue blocks will then be imaged with serial sectioning polarization sensitive optical coherence tomography (PS-OCT) to obtain 20 μm isotropic resolution images of cyto- and myelo-architectural features and fiber tractography. Registration with the MRI is preserved because imaging is performed before sectioning. Serial sections with 150 μm thickness are cut from the block face, cleared, and subsequently imaged by selective plane imaging microscopy (SPIM) with 1 μm isotropic resolution, and with multiplexed histological staining of multiple molecular markers that will facilitate cellular classification. Segmentation of common features amongst the SPIM, OCT, and MRI, permits registration of the cellular information from SPIM back to the MRI coordinate system with OCT serving as an important intermediary permitting registration of SPIM with MRI with micrometer resolution. Our aims are designed to establish our imaging workflow and build a cell census atlas for language regions in human frontal cortex, resulting in a clear path forward for obtaining the cell census for the entire human brain.</div><div class = "text-block">For this protocol, we will be focusing at the data acquisition and imaging processing of serial sectioning PSOCT.</div></div>

[STEPS]