eamag/protocols-with-steps huggingface dataset:
TITLE: Bulk tissue RNA-sequencing - hiPSC-derived astrocytes, neurons and co-cultures 
AUTHORS: Karishma D'Sa

[DESCRIPTION]
Bulk tissue RNA-sequencing of hiPSC-derived astrocytes, neurons and co-cultures using the Illumina TruSeq Stranded mRNA Library Prep kit

[STEPS]
SECTION: Bulk tissue RNA-sequencing
1. Libraries for sequencing were prepared using the Illumina TruSeq Stranded mRNA Library Prep kit by loading 50 ng of total RNA into the initial reaction; fragmentation and PCR steps were undertaken as per the manufacturer’s instructions. 
SECTION: Bulk tissue RNA-sequencing
2. Final library concentrations were determined using Qubit 2.0 fluorometer and pooled to a normalized input library. 
SECTION: Bulk tissue RNA-sequencing
3. Pools were sequenced using the Illumina NovaSeq 6000 Sequencing system to generate 150 bp paired-end reads with an average read depth of ~137 million paired-end reads per sample.
TITLE: Media Preparation for E. coli growth for Cell-free Protein Synthesis
AUTHORS: Weston Kightlinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cell-free protein synthesis reactions require cell-free extract derived from rapidly dividing E. coli cells to produce high yields of protein therapeutics, antigens, enzymes, vaccines, and other proteins. Standard protocols use 2xYTPG media for cell growth. This protocol is for making 1 L of 2xYTPG media.</div><div class = "text-block"><span>Read more on cell-free protein synthesis here: </span><a href="https://www.swiftscalebio.com/blog/cell-free-protein-synthesis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.swiftscalebio.com/blog/cell-free-protein-synthesis</span></a></div></div>

[STEPS]
?. [Media Preparation for E. coli growth for Cell-free Protein Synthesis]
Mix ingredients except for glucose with water below the amount of volume of MilliQ water needed using a plastic beaker and a stir-bar. Once dissolved (you can use a spatula to help break up clumps), pour into a big plastic graduated cylinder and adjust to final volume then mix in original beaker then disperse  to  Tunair flasks for autoclaving.
750 mL
2.5 L
?. [Media Preparation for E. coli growth for Cell-free Protein Synthesis]
Prepare the glucose separately in individual  containers and add just before inoculation.
250 mL
?. [Media Preparation for E. coli growth for Cell-free Protein Synthesis]
Calibrate pH probe.
?. [Media Preparation for E. coli growth for Cell-free Protein Synthesis]
pH the 2xYTP solution to  using 5 N KOH.
Expected Result: Expected concentration of protein in Cell-free extract is ~  so make the following dilutions:
TITLE: Assessing the post-nuclear testing genetic health of Pacific Islanders on Mo&#39;orea, French Polynesia
AUTHORS: Kedhar Bartlett

[DESCRIPTION]
The atrocities of imperial nuclear testing in the Pacific Ocean have gone without widespread recognition or addressing for too long. Health effects caused by radiation exposure can be screened for and examined genetically. The objective of this project is to collect data regarding the genetic health of ethnic Pacific Islanders on the island of Mo’orea; this data will be used to properly assess the lasting genetic and genomic effects of nuclear testing on Pacific Islanders, and to facilitate genetic counseling to individuals at no cost.

[BEFORE_START]
Participants will have been contacted randomly following a public outreach campaign assisted by local government and cultural leaders. Those interested will fill out a form.

[STEPS]
SECTION: Collecting DNA Sample
3. Lay out all materials
SECTION: Sequencing DNA
6. Isolate DNA
SECTION: DNA Analysis
9. Paste DNA sequence into NCBI BLAST software
SECTION: Contacting Participants
1. Contact participant by email and (if phone number provided) phone, attempting to set up a time and location to meet for data collection
SECTION: Contacting Participants
2. Confirm date, time, and location at least one week in advance. Record on team calendar.
SECTION: Collecting DNA Sample
4. Explain to patient how to perform the buccal swab
SECTION: Collecting DNA Sample
4.1. Unwrap buccal swab and ensure the collection end does not touch anything but the inside of your cheek
SECTION: Collecting DNA Sample
5. Stir swab around lysis solution (breaking membranes, first step to isolating DNA) 
SECTION: Collecting DNA Sample
4.2. Gently swab the inside of both cheeks, then "scrape" the inside of both cheeks
SECTION: Collecting DNA Sample
3.1. Put lysis solution in test tubes
SECTION: Collecting DNA Sample
4.3. Return swab to technician
SECTION: Sequencing DNA
6.1. Pour the contents of the test tube into centrifuge tubes
SECTION: Sequencing DNA
6.2. Follow lab procedure to isolate DNA
SECTION: Sequencing DNA
7. Perform PCR
SECTION: Sequencing DNA
7.1. Thermal cycler is automatic; place centrifuge tubes in thermal cycler and let run
SECTION: Sequencing DNA
8. Put amplified DNA in sequencer and allow text file to download to computer
SECTION: (Optional) Genetic Counseling
12. If concerning results are found with regards to patient health outlook, gently inform the patient and give them our informational pamphlet on genetic counseling services offered.
SECTION: DNA Analysis
10. Run comparative analyses with pre-selected positive controls for radiation-related diseases such as thyroid and colon cancer, and other common radiation mutagen-associated genetic conditions
SECTION: (Optional) Genetic Counseling
13. Answer any questions they may have and direct to resources (hospitals, specialists, etc.) on the pamphlet.
SECTION: DNA Analysis
11. Record data in sheet provided (positive or negative for each condition, as well as specific marker indication for conditions that are associated with multiple mutations)
TITLE: A Randomized Trial of a Pilot Behavioral Support Intervention After Bariatric Surgery
AUTHORS: Michelle Lent

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bariatric surgery patients may experience significant psychosocial changes after surgery, but little</div><div class = "text-block">psychological support is available beyond support groups postoperatively. We evaluated the feasibility of a</div><div class = "text-block">postoperative support program targeting quality of life, psychosocial functioning and adherence to behavior change in bariatric surgery patients. This prospective, randomized pilot trial evaluated a comprehensive postoperative behavioral support intervention using a 4-month bi-weekly program in 24 bariatric surgery patients compared to 26 usual care patients that completed bariatric surgery within one year. Outcomes included feasibiilty and the difference in quality of life (as measured by the Short Form-36) between groups, as well as differences in psychosocial functioning (mood, eating behaviors) and adherence (diet, physical activity, appointments). Outcomes were assessed at baseline and treatment completion (4 months). The intervention focused on addressing psychosocial changes after surgery, strategies for postoperative diet and adherence, and preventing weight regain. Patients collaboratively set goals for diet, physical activity, adherence and other behavioral changes tailored to the needs of each participant. Intervention patients were asked to attend 8, one-hour bi-weekly treatment sessions in a 4-month time period.</div></div>

[STEPS]
TITLE: NCEM Drop - Tissue Dounce Homogenisation (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
cell pellet dounce homogenisation

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Conventional
9. Adsorb 10 µL sample to grid 10 min , inspect to ensure sample does not dry out.

SECTION: Conventional
10. Drain excess sample from grid using filter paper, leave wet.
SECTION: Conventional
11. Stain  1 min 

12. Drain & dry using filter paper
SECTION: Dounce Homogenisation
2. Remove cells from the flasks by scraping with a disposable plastic scraper and poor into a 10 mL centrifuge tube. 

SECTION: Dounce Homogenisation
5. Insert teflon plunger into cordless drill, set drill on max speed.
SECTION: Dounce Homogenisation
7. Clarify resultant solution at 13000 rpm  in Eppendorf centrifuge for 1 min 


SECTION: Dounce Homogenisation
8. Stand for 5 min to permit viruses to diffuse back into solution, from the debris, and provide an interface for the sampling of membrane associated viruses. Use supernatant for sample below.

SECTION: Dounce Homogenisation
3. Pellet the cells in a bench top centrifuge at 2000 rpm 2 min , remove supernatant.

SECTION: Dounce Homogenisation
4. Resuspend the cells in equal volume TC water and transfer to glass homogeniser tube.
SECTION: Dounce Homogenisation
6. Homogenise(20 strokes) inside Class II BSC cabinet.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>
<p> </p>
<p>Note: For Human CD4 Nanobeads, please refer to the protocol found on the product <a href="http://www.biolegend.com/media_assets/support_protocol/MojoSort_Human_CD4_Nanobeads_Protocol_V02_10192015.pdf" target="_blank">webpage</a> for MojoSort™ Human CD4 Nanobeads (Cat. No. 480013/480014).</p>
<p> </p>

[GUIDELINES]
<p>This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p><br />-MojoSort™ Buffer (5X) (Cat. No. 480017)<br />-MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />-Adjustable pipettes<br />-70 μm filters (one per sample)<br />-5 mL (12 x 75 mm) polystyrene tubes<br />-Reagents for sample preparation<br />-Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><strong>Application notes</strong>: To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dnwecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dnvecje.jpg" data-ofn="MojoSort.JPG" /></p>
<p> </p>

[STEPS]
?. 
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?.
TITLE: Post mortem human substantial nigra TH staining
AUTHORS: Waijiao Kai, Xiqun Chen

[DESCRIPTION]
This protocol is standardized for postmortem (frozen) SN tissue from pathologically diagnosed PD patients and control individuals for Immunofluorescence staining. 

[STEPS]
1. Deparaffinization
Wash slides in 2 changes of 300 mL ml of xylene – 6 min each at room temperature.

2. Rehydration
Wash slides in 300 mL of 100% alcohol –9 min at Room temperature.
Wash slides in300 mL of 95% alcohol – 6 min at Room temperature.
Wash slides in 300 mL of 80% alcohol – 3 min at Room temperature.
Rinse slides in gentle running distilled water – 5 min atRoom temperature.

3. Antigen retrieval
Microwave (high power) slides in 200 mL 0.01 Molarity (M) sodium citrate buffer, pH 6.0  at 99-100 °C for 5 min, three rounds. 
Add 40 mL distill water after 1st round
Add 150 mLsodium citrate buffer after the 2nd round.
Let stand at room temperature in the buffer - 20 min
Rinse in 1X TBS with Tween (TBST) – for 5 min at -100 Room temperature.

4. Immunostaining
Do not allow tissues to dry at any time during the staining procedure.
Apply a universal protein block (5% goat serum in TBST) – for 30 min at -100 Room temperature.
Drain protein block from slides, and apply diluted primary antibody overnight at 4 °C.
Rinse slides in 1X TBST 3 times - 5 min each at -100 Room temperature.
Incubate secondary antibody  (Conc  - 1:400) - 30 min at 37 °C .
Rinse slides 1X TBST 3 times – 5 min each at -100 Room temperature
Mount the slides with ProLong Gold antifade
TITLE: Measurement of Left Ventricular Hemodynamic Parameters in Intact Mice
AUTHORS: E. Dale Abel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes the procedure used by the DiaComp for measuring left ventricular hemodynamic parameters in intact mice.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bihs4kcn.jpg" /></div></div>

[STEPS]
?. Measurement of in vivo cardiac contractile performance using a micro-manometer tipped catheter:
Indices of left ventricular contractile performance are determined in mice under isoflurane anesthesia by percutaneously introducing a 1.4 Fr. Millar catheter into the carotid artery and gently advancing this into the left ventricular cavity. Pressure waves are recorded captured and analyzed with pClamp 8.2 software. This technique provides the following indices of left ventricular function.(1)  Left ventricular systolic pressure (LVSP) Units: mmHg
(2)  Left ventricular end diastolic pressure (LVEDP) Units: mmHg
(3)  Heart Rate Units: beats per minute
(4)  Positive dP/dt – First derivative of LV pressure. This index is a measure of the rate of pressure development in the left ventricle. Units: mmHg/msec.
(5)  Negative dP/dt – First derivative of LV pressure. This index is a measure of the rate of ventricular relaxation, or the rate of pressure decay in the LV Units: mmHg/msec.
TITLE: Salmonella blood culture surveillance: SOP&#39;s ITM
AUTHORS: Bieke Tack

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standard operating procedures for Salmonella blood culture surveillance of the unit of tropical bacteriology at the Institute of Tropical Medicine in Antwerp (Belgium):</div><div class = "text-block">- Blood culture work-up</div><div class = "text-block">- Pathogen identification</div><div class = "text-block">- Salmonella serotyping</div><div class = "text-block">- Antibiotic susceptibility testing</div></div>

[STEPS]
TITLE: Mouse tissue collection
AUTHORS: Tae-Un Han

[DESCRIPTION]
This protocol is about collecting mouse tissues for immunohistochemistry or biochemistry experiment. While it is mainly used for brain tissues, it also can be used for other visceral tissues. 

[STEPS]
SECTION: Perfusion
1. Mice are anesthetized with Avertin (1mL/25g body weight).
SECTION: Perfusion
7. Tissues are collected and post-fixed in 4 % volume PFA at 4°C for about 1440 min

SECTION: Perfusion
2. Immobilize a mouse and cut through the skin and muscles in the lower abdomen exposing the liver using a sharp scissors.
SECTION: Frozen tissues
11. Tissues for biochemical experiment such as gene expression analysis, protein assay or lipidomics are collected using snap-freezing in dry ice without perfusion, then stored at -80 °C  o C.

SECTION: Perfusion
4. Grasp the heart firmly near the apex with a blunt forceps, then insert the butterfly needle gently into the left ventricle.
SECTION: Perfusion
3. Cut through the diaphragm, then down the right and left sides of the chest, through the ribs, until the heart is fully visible.
SECTION: Perfusion
5. Using a small, sharp scissors, make a small cut in the right atrium to serve as a drain.
SECTION: Perfusion
6. Flow 1x PBS (Quality Biological) containing 1 IU Heparin/mL (about 30 s) followed by 4 % volume paraformaldehyde for fixation until evidence of stiffness of tissues is observed.

SECTION: Perfusion
9. The floating sections are collected with microtome combined with freezing unit. After getting 30~40 um thickness of floating sections, they can be stored in PBS-sodium azide (0.05 % volume ) solution for 80640 min  maximum.

SECTION: Perfusion
8. For floating sections, tissues are transferred to 30% sucrose for cryoprotection, then kept at 4 °C for about 2880 min until tissues sink to the bottom of the vessel.

SECTION: Perfusion
10. For paraffin sections for histopathology evaluation, tissues are transferred to 70 % volume ethanol after fixation then kept at 4 °C  until usage.
TITLE: Cell Fixation and Permeabilization Protocol using 70% Ethanol
AUTHORS: Sam Li

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

[STEPS]
?. Prepare 70% Ethanol and chill to -20°C.Tip: Do not freeze ethanol for long-term storage.
?. Prepare target cells of interest and wash 2X with PBS, centrifuging at 350xg for 5 minutes
?. Discard supernatant and loosen the cell pellet by vortexing.
?. Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.
?. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.
?. Wash 2X with BioLegend Cell Staining Buffer (Cat. 420201) and resuspend cells at 0.5-1.0 x 107 cells/ml.
?. Use 100µl cell suspension/staining tube.Note: Please note that certain markers or fluors may not survive ethanol fixation. Protein-based fluors, like PE and APC, tend to have more difficulty, while synthetic fluors, like Brilliant Violet™, tend to have a higher chance of surviving the process.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression. </p>

[BEFORE_START]
<div title="Page 1">
<p><strong>Checklist prior to beginning:</strong><br />-Centrifuges, large and small, set to 4 ºC</p>
<p>-Make enzyme stock; thaw two aliquots of <em>Bacillus Licheniformis</em> enzyme on ice.</p>
<p>-Make 0.04% BSA/PBS (50 mL)<br />-Things you need: petri dishes, clean forceps, razor blade, pipets, 30 μM filters, timer.</p>
<p> </p>
<p><strong>Stock solution for enzyme </strong></p>
<ul>
<li>895 μL DPBS</li>
<li>5 μL 0.5 M EDTA (2.5 mM final)</li>
</ul>
<p>→Add 100 μL enzyme (100 mg/mL) to 900 μL of enzyme stock to make 1X enzyme mix. Add 28 mg of tissue to each 900 μL of enzyme mix.</p>
</div>

[GUIDELINES]
<table>
<tbody>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p><strong>Reagent</strong></p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<p><strong>Storage Condition </strong></p>
</div>
</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>DPBS (Thermofisher, 14190144)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<p>4°C</p>
</div>
</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>0.5 M EDTA (Ambion, AM9260G)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<div title="Page 1">
<div>
<div>
<div>
<p>room temp.</p>
</div>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>BSA (Sigma, A8806)</td>
<td>4°C</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>Protease from <em>Bacillus Licheniformis </em>(Sigma, P5380)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C</p>
</div>
</div>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><a href="https://www.creative-biolabs.com/car-t/cellrapeutics-chimeric-antigen-receptor-car-technology.htm" target="_blank" rel="noopener noreferrer">CAR-T therapy</a> plays a key role in cancer immunotherapy and belongs to adoptive T cell transfer (ACT). At the beginning of June, the annual meeting of the American Society of Clinical Oncology (ASCO) was held in which CAR T therapy was still a hot topic. And Celgene and Bluebird have released the latest cell therapy data, showing once again the potential of this new cancer immunotherapy.</p>
<p> </p>
<p>In 2017, the first two CAR-T cell therapies approved by FDA—Kymriah of Novartis and Yescarta of Kite Pharma (acquired by Gilead) were used to treat acute lymphoblastic leukemia and specific types of non-Hodgkin’s lymphoma, respectively, contributing to the rapid development of CAR-T therapy.</p>
<p> </p>
<p>CAR-T therapy is very different from traditional drugs. First, the therapy requires the isolation of T cells from the patient's body. The T cell is modified <em>in vitro</em> using a <a href="https://www.creative-biolabs.com/car-t/cellrapeutics-chimeric-antigen-receptor-car-technology.htm" target="_blank" rel="noopener noreferrer">chimeric antigen receptor</a> (CAR) to specifically recognize cancer cells, and then the transformed T cells are expanded and returned to the patient's body.</p>

[STEPS]
?.
TITLE: Preparation of B cells for scRNAseq
AUTHORS: Nicholas Pease

[DESCRIPTION]
This protocol details activated B cell preparation and sample hashtagging for Chromium Single Cell  Gene Expression (10x Genomics).

[STEPS]
SECTION: Dead cell removal
1. Pellet cells at 300xg for 10min
SECTION: Dead cell removal
2. Resuspend in 100uL Dead Cell Removal Buffer and filter through 40uM cell strainer
SECTION: Dead cell removal
3. Add 5uL of Annexin V Cocktail to sample
SECTION: Dead cell removal
4. Add 5uL of Biotin Selection Cocktail to sample, mix and incubate at RT for 3min
SECTION: Dead cell removal
5. Vortext RapidSpheres for 30 sec and add 10uL to each sample, flick to mix
SECTION: Dead cell removal
6. Add 2.4mL of Dead Cell Removal buffer and mix again
SECTION: Dead cell removal
7. Place tubes on magnet and incubate at RT for 10 min
SECTION: Dead cell removal
8. Transfer supernatant to new 15mL tube
SECTION: Dead cell removal
9. Aliquot
SECTION: Sample hashtagging
10. Resuspend samples in 100uL of wash buffer (PBS + 10% FBS)
SECTION: Sample hashtagging
11. Add 1uL of HTO antibodies
SECTION: Sample hashtagging
12. Incubate for 30 min on ice
SECTION: Sample hashtagging
13. Add 3.5mL of wash buffer to labeled cells, mix and pellet at 200xg for 10min at 4C
SECTION: Sample hashtagging
14. Remove supernatant with pipette, resuspend pellet with 3.5mL of wash buffer, and transfer to new tube
SECTION: Sample hashtagging
15. Pellet at 200xg for 10min at 4C
SECTION: Sample hashtagging
16. Remove supernatant with pipette and resuspend pellet with 3.5mL of wash buffer
SECTION: Sample hashtagging
17. Pellet at 200xg for 10min at 4C
SECTION: Sample hashtagging
18. Assuming 50% cell loss, resuspend pellet in wash buffer to obtain ~1,000 cells/uL
SECTION: Sample hashtagging
18.1. Count cells and adjust concentration, if needed
SECTION: 10x loading
19. Mix hashtag samples at desired ratio and load 52,500 cells per well
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The diversity of ribonucleic acid (RNA) viruses in the ocean and the ongoing isolation and characterization of RNA viruses that infect important primary producers suggests that RNA viruses are active members of the marine microbial assemblage. At this point, little is known about the composition, dynamics, and ecology of the RNA virioplankton. In this collection, we describe two methods to assess RNA virus diversity from seawater.

[STEPS]