eamag/protocols-with-steps huggingface dataset:
TITLE: Tranformation of Thalassiosira pseudonana via bacterial conjugation
AUTHORS: Ana Cristina Jaramillo Madrid, Justin Ashworth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol has been successfully used to express nourseothricin resistance gene, mVenus fluorescence protein and other proteins related to triterpenoids production in Thalassiosira pseudonana (Tp) strain </span><a href="https://ncma.bigelow.org/ccmp1335?___SID=U#.WAQoEvkrL4Y" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">CCMP1335</span></a><span>.  The original protocol was published by Karas et al. (2015) where a  detailed description of L1 medium and plates preparation is presented. </span></div></div>

[STEPS]
?. [Growth and preparation of E. coli donor]
Inoculate 5 mL LB medium (gentamicin+antibiotic 2) with bacterial colonies from the gentamicin +antibiotic 2 plates. Grow overnight.
?. [Growth and preparation of E. coli donor]
Start a 150 mL LB subculture with the 5 mL overnight culture (recommended starting OD600 either 0.05 or 0.1).
?. [Growth and preparation of E. coli donor]
Grow at 370C until OD600 reaches 0.4. (A range of OD600  from 0.4 to 0.6 has worked for me)
?. [Growth and preparation of E. coli donor]
Centrifuge at 4000 g, 100C, for 10 min.
?. [Growth and preparation of E. coli donor]
Decant supernatant and resuspend in 800 µL SOC.
?. [Growth and preparation of diatom cells]
Measure the Thalassiosira pseudonana cell concentration and calculate the required volume needed to collect 2 x 108 cells. Tp cells are cultured in L1 medium. Note: We do not know if cell density before spinning cells down matters. We have successfully tried spinning cells down at ~2-4 x 106 cells/mL
?. [Conjugation]
Mix 200 µL diatom cells and 200 µL E. coli cells in a 1.5 mL tube.
?. [Conjugation]
Pipette up and down a few times.
?. [Conjugation]
Plate on 1/2xL1 1% agar plates w/ 5% LB. Note: Make sure the plates are dry.
?. [Conjugation]
Incubate in dark at 300C for 90 minutes.
?. [Conjugation]
Move plates to standard diatom growth conditions. Incubate 20-24 hrs Note: Supposed to be 180C and constant light, but we just leave them at RT constant light.
?. [Growth and preparation of diatom cells]
Spin down 4000 g, 10oC, for 10 min
?. [Selection]
Add 1 mL L1 medium and scrape.
?. [Selection]
Plate 200 µL of the resulting suspension on pre-dried 1/2xL1 1% agar plates w/ 50 ug/ml nourseothricin.
?. [Selection]
Leave at 18oC and constant light until colonies appear in ~10-12 days.
?. [Growth and preparation of diatom cells]
Decant supernatant and resuspend pellet in 1 mL L1 medium. Final concentration 2 x 108 cells/m
TITLE: Final QC, Pooling and Sequencing 
AUTHORS: Katarina A Cohen, Khai-Minh H Nguyen, Oksana Polesskaya, Abraham Palmer

[DESCRIPTION]
This protocol is conducted after a set of libraries are completed and ready to quantify and pool. This protocol outlines the final steps before submitting for sequencing. 

[BEFORE_START]
Ensure all libraries that will be pooled are uniquely indexed. 

[STEPS]
SECTION: Library QC
1. Quantify purity and concentration of library with Nanodrop and a Qubit Assay
Obtain average fragment size of library with Tapestation (D1000 Assay) 




SECTION: Pooling
2. Download   
SECTION: Pooling
3. 
Increase "Target Vol (uL) per Sample" if any Sample Vol is lower than 1ul
Use   when adding water to final pool 

SECTION: Checking Pooling
9. RECOMMENDED OPTIONAL STEP
Check pooling with an illumina MiSeq run 
% Reads Identified for each library shouldn't vary more than 30% from each other. 
SECTION: Sample-Barcode List
10.   
Download the file above
Check Twist Bioscience site for updates to the sample barcodes used for the Twist 96-Plex Kit. 




SECTION: Sample-Barcode List
12. When the "Sample_Randomization" tab is opened, copy and paste the randomized Transponder ID's into Column A of the Flowcell Sample-Barcode list.

NOTE: If less than 96 samples are processed in a  library, delete the unfilled rows within that 96 library set. (Essentially, you want to delete the sample barcodes that are not associated with a sample). 


SECTION: Sample-Barcode List
11. Open the library file created in the "EPMotion - Normalization and Randomization". 
Go to the "Sample_Randomization" Tab
SECTION: Sample-Barcode List
13. Transfer any comments from the library file into Column G of the Sample barcode list
Ensure that the comment matches with the correct sample ID. 
SECTION: Sample-Barcode List
14. Enter the PCR index barcode used for that particular library (ex. 1,2,3,4,5...) in Column D
SECTION: Sample-Barcode List
15. Enter Library name in Column E 
SECTION: Submitting for Sequencing
16. Submit 30ul of Pool 
Platform: NovaSeq S4
Run Type: PE150

You can find the updated inline i7 and i5 index sequences on Twist Bioscience Site 

SECTION: Library QC
1.1. Libraries should have an average fragment size between 420bp - 650bp.
260/280 should be around 1.80 - 2 
260/230  should be around 2-2.2.
We have been able to get good data from libraries with relatively poor nanodrop purities.
Qubit concentrations can widely range. We get a range from 10ng/ul - 60ng/ul 

SECTION: Pooling
4. Enter library name in Column A "Sample Name"
SECTION: Pooling
5. Enter qubit concentrations in column B
SECTION: Pooling
6. Enter average fragment size of library in column C for each library. 
SECTION: Pooling
7. Increase "Target Vol (uL) per Sample" if any Sample Vol is lower than 1ul
SECTION: Pooling
8. Label new tube as Riptide Pool ## , and Date. Add calculated volume of water (shown in cell I6). Add calculated volumes of sample shown in column E. 
Use   when adding water to final pool
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Must be made fresh before experiment because of the Sucrose. <br />For 20 mL solutions.

[BEFORE_START]
Prepare solution with sterile milliQ water and filter sterilize or autoclave before use.

[GUIDELINES]
Final Concentration of Lysis Buffer<br />40 mM EDTA<br />50 mM Tris (pH 8.3)<br />0.73 M Sucrose

[STEPS]
?. 
?. 
?.
TITLE: Pneumonia Associated with Invasive and Noninvasive Oxygenation Strategies for Acute Hypoxemic Respiratory Failure in Adults: A Systematic Review and Network Meta-analysis Protocol
AUTHORS: Satoshi Hokari, Shunsuke Kimata, Masaaki Sakuraya, Hiromu Okano, Tomoyuki Masuyama

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Background: Noninvasive oxygenation strategies, such as noninvasive positive pressure ventilation and high-flow nasal cannula, have become common treatments for acute hypoxemic respiratory failure. However, further research is needed to understand the relative benefits and risks of each strategy.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Aim: The purpose of this systematic review is to assess which noninvasive oxygenation strategy is best for reducing pneumonia in patients with acute hypoxemic respiratory failure.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Methods and analysis: We will conduct a systematic review of the relevant literature according to the Cochrane Handbook and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. We will include randomized controlled trials assessing the effect of noninvasive oxygenation interventions for patients with acute hypoxemic respiratory failure. The main outcome is the incidence rate of pneumonia, which is not limited ventilator-associated pneumonia, aspiration pneumonia, and nosocomial pneumonia. Two independent reviewers will extract the data and assess the risk of bias. We will perform a meta-analysis using the GRADE Working Group Approach for a network meta-analysis.</div></div></div>

[STEPS]
TITLE: Thawing and Seeding Frozen Cells
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to thaw cells from the liquid nitrogen storage and seed into a tissue culture flask</div></div>

[STEPS]
?. [Thaw Cells]
Thaw cells by suspending cryotube in  water bath until completely thawed, but no longer than necessary
?. [Transfer cell suspension]
Within biosafety cabinet, transfer cell suspension to 15 mL centrifuge tube using 1000 μL pipette.
?. [Dilute freezing medium]
Add  warmed cell culture medium to cell suspension dropwise.
1 ml
Adding the inital cell culture medium slowly helps prevent cell death caused by a rapid change in osmotic pressure.
?. [Dilute freezing medium]
Add an additional  warmed cell culture medium to cell suspension slowly.
3 ml
?. [Centrifuge cell suspension]
Centrifuge the cell suspension at  for .
?. [Resuspend Cells]
Remove bulk of supernatant with serological pipet, then remove remainder with 1000 μL pipette.
For small cell pellets, you are better off leaving a small amount of media than disturbing the cell pellet.
?. [Resuspend Cells]
Add  warmed cell culture media to cell pellet.
1 ml
Allowing the cell pellet rest in media for about 2 minutes will help with resuspension.
?. [Resuspend Cells]
Gently pipette mix the cell pellet into the solution.
?. [Resuspend Cells]
Add an additional  warmed cell culture media [ for T-25].
7 ml
3 ml
?. [Seed Cells]
Using a serological pipet, transfer the cell suspension to the tissue culture flask.
?. [Label Flask]
Label the flask with:Cell linePassage numberDateYour intials
?. [Incubate]
Transfer flask to CO2 incubator.
?. [Documentation]
Don't forget to remove the vial you used from the frozen storage inventory.
TITLE: Regular maintenance of human pluripotent stem cells
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the regular maintenance and passaging human iPSCs.

[STEPS]
SECTION: Day 1
1. Pre coat a 6 well dish with Matrigel matrix for 1440 min or 1 hour.

SECTION: Day 1
2. Thaw the frozen iPSCs by placing the vial in 37 °C water bath for 2 min.

SECTION: Day 1
3. After thawing, spray the tube with 70% ethanol and place in biosafety cabinet.
SECTION: Day 1
4. Aspirate the cells into 15 ml falcon tube and add 4 mL cold E8 media containing rock inhibitor.

SECTION: Day 1
5. Spin down the tube at 0.3 rcf, 3 min.

SECTION: Day 1
6. Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
SECTION: Day 1
7. Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
SECTION: Day 2
8. Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
SECTION: Day 2
9. Feed every day with fresh media till plate gets 60-70% confluency.
SECTION: Day 2
10. Cells were passaged every 5 days with E8.


SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
11. Pre coat a 6 well dish with Matrigel matrix for 1440 min or 1 hour.

SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
12. Remove the culture media.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
13. Rince 1X with sterile PBS.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
14. Add 1 mL of EDTA solution.

SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
15. Place in the incubator for 5-7 minutes.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
16. Bring the plate into biosafety cabinet and add 2 mL E8 media containing rock inhibitor.

SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
17. Dispense all the cell suspension into 15 ml falcon tube.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
18. Spin down the tube at 0.3 rcf, 3 min.

SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
19. Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 1
20. Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 2
21. Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
SECTION: iPSC passaging with 0.5 mM EDTA solution: Day 2
22. Cells were passaged every 5 days with E8.
TITLE: U Mass - Aspartate Transferase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of aspartate transferase reflect liver function.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Aspartate Transferase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: cDNA Library Preparation for scRNA-seq of Human Meniscus (10x Genomics) 
AUTHORS: molmer, Martin Lotz, Tony Mondala, Steven Head

[DESCRIPTION]
Samples are processed using V2 barcoding chemistry kits of 10x Genomics. For each run, 10,000 cells from individual donors are labeled with distinct oligo-barcoded antibodies (cell hashing), enabling us to pool samples from both conditions on each 10X Genomics run and reduce batch effects. All samples from a given experiment are processed in parallel in the same thermal cycler. The 10x user guide can be found https://assets.ctfassets.net/an68im79xiti/1C16trEdzy1Folq5xbOijE/7e6fb1f504e130bd561d898384da99d9/CG000315_ChromiumNextGEMSingleCell3-_GeneExpression_v3.1_DualIndex__RevB.pdf and is also an attached document.

[STEPS]
1. Single cells are isolated from human knee menicus using doi dx.doi.org/10.17504/protocols.io.n2bvj6k7blk5/v1.
2. Single cell suspensions are multiplexed and converted to barcoded scRNAseq libraries using the Chromium Single Cell 3’ Library, Gel Bead and Chip Kit (10x Genomics), and the BD™ Hu Single Cell Sample Multiplexing Kit.
 


3. Libraries are sequenced on an Illumina NextSeq2000 sequencer targeting 20,000-25,000 reads per cell.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Clean the work area with 70% alcohol. Use all filters and autoclaved tips. Refrigerate your centrifuge to 4C.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: CoBG-11 preparation
AUTHORS: Arshshaikh  

[DESCRIPTION]
CoBG-11 is a coculture medium used to coculture cyanobacteria and E. coli by Zhang et al (2020). It is optimized for E. coli growth. Here are its components:

1. 150 mM NaCl,
2. 4 mM NH4Cl
3. 3 g/L 2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl] amino] ethanesulfonic acid (TES)

The pH value is adjusted with NaOH to 8.3.

The protocol below describes the preparation of 100ml of coBG-11. 

[STEPS]
1. Measure 0.88 g , 0.0214 g, and 0.3 g and add it to a 150mL flask.
 

 

2. Add 50 mL to the flask and mix till contents dissolve. If salt persists, add 10mL more of BG-11 and mix well.

3. Once contents dissolve, add BG-11 up to 95 mL.
4. Adjust pH of the solution to 8.3 with NaOH.
5. Fill BG-11 up to 100 mL measure pH and adjust if required.
TITLE: SOLUTION- 08 - Türk solution
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recepe is used in the following protocols:</div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01a - </span><span>Isolation of Human PBMC from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 01b - </span><span>Isolation of Human PBMC from Whole Blood</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 02 - </span><span>CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PBMC- 03 -</span><span> TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01a - </span><span>Isolation of Human PMN from Buffy Coat</span></div><div class = "text-block"><span style = "font-weight:bold;">- PMN- 01b - </span><span>Isolation of Human PMN from Whole Blood</span></div></div>

[STEPS]
TITLE: Triage for critically ill patients under the limited medical resource capacity during the COVID-19 and other infectious disease pandemics: a scoping review.
AUTHORS: Yusuke Tsutsumi, Yasushi Tsujimoto, Sei Takahashi, Asuka Tsuchiya, Ikuyo Tsutsumi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Introduction </div><div class = "text-block">COVID-19 spread worldwide rapidly since December 12, 2019, affecting more than 180 countries. The rapidly increased number of patients sometimes exceed medical resource capacity. Under the scarce resource situation, clinicians may need to allocate medical resources to selected populations. However, there are no established triage criteria. Therefore, we conducted a scoping review to map the reported triage available. We formulated the following primary review question:  What kind of criteria and algorithms are available for clinicians to triage patients in the resource scarcity setting during the disease pandemic like COVID-19?</div><div class = "text-block">Methods and analysis </div><div class = "text-block">For conducting the scoping review protocol, we will use a scoping review methodology established in the Joanna Briggs Institute manual. We also will use the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews to assess the results. The concept of our review will be the triage criteria, systems, or algorithms used for the allocation of medical resources to selected populations among patients with the pandemic disease. The context of our review will be the resource scarcity setting during disease pandemic like COVID-19. We will conduct the systematic search on MEDLINE, Embase, and Cochrane controlled register of trials (CENTRAL) and include studies that incorporate critically ill patients due to the pandemic disease itself. We will summarize the studies based on the characteristics and efficiency (if applicable) of triages.</div><div class = "text-block">Ethics and dissemination </div><div class = "text-block">Since this is a review of the literature, ethics approval is not mandated. We will publish the results from this review in peer-reviewed journals.</div></div>

[STEPS]
TITLE: General Salts + Sodium Bicarbonate Solutions
AUTHORS: Ada de la Cruz

[STEPS]
?. [General Salts Solution:]
For 1L of 10x salts solution:1 L nanopore water into 1 L bottle with lid55 g of MgCl2*6H2O5 g of NH4Cl1.4 g CaCl2*2H2O1 g KCl
?. [Sodium  Bicarbonate]
For 1 L of NaHCO3 (sodium bicarbonate):1 L nanopore water in a 1 L flask/bottle85 g of NaHCO3
?. [General Salts Solution:]
Pour everything into a beaker, place a magnetic stir bar inside, place on top of stir plate and turn on. The salts should immediately go into solution.
?. [Sodium  Bicarbonate]
Pour into a beaker, place a magnetic stir bar inside, place on top of stir plate and turn on. It should go into solution. HOWEVER...
?. [Sodium  Bicarbonate]
If NaHCO3 does not go into solution, add 4M HCl in small increments until NaHCO3 eventually dissolves.If you've added a lot of HCl and it still does not go into solution, turn stir plate off, let sit for 10 minutes, and turn back on (this sometimes helps it go into solution).
?. [General Salts Solution:]
Place lid on bottle, label, and place into fridge.
?. [Sodium  Bicarbonate]
Pour solution into small flasks in 25 mL or 50 mL amounts until all 1000 mL have been dispensed. Seal all flasks with a stopper.
?. [Sodium  Bicarbonate]
Place flasks into an autoclave bin, place autoclave tape on them, and autoclave (P12 30 min. liquid setting)
TITLE: HuBMAP Donor and Tissue Eligibility Criteria Form v 1.0
AUTHORS: Yiing Lin, Shin Lin

[STEPS]
?. HuBMAP Donor and Tissue Eligibility Criteria FormVersion 1.0Effective Date: 1/1/2019                                                                                                                                Ineligible            EligibleOverall eligibilityDoes the consent allow for research activities?                                            ☐ No                    ☐ YesCan the tissue collection be completed within 4 hours of clamp time?    ☐ No                    ☐ YesIs the HIV/HCV/HBV NAT non-reactive?                                                         ☐ No                    ☐ YesIs the HBV Surface Antigen test non-reactive?                                              ☐ No                    ☐ YesIs the HCV Encoded Antigen test non-reactive?                                            ☐ No                    ☐ YesFor collection of bowel samplesIs there a prior history of gastrointestinal disease?                                     ☐ Yes                  ☐ NoDoes the CT scan show any evidence of bowel abnormality?                   ☐ Yes                   ☐ NoFor collection of heart samplesIs the EKG normal?                                                                                            ☐ No                    ☐ YesIf done, does the echocardiogram show EF >= 50%?                                  ☐ No                    ☐ Yes / not doneFor collection of lung samplesDoes the CT scan note lung pathology such as bullae or pneumonia?   ☐ Yes                   ☐ NoFor collection of pancreas samplesDoes the CT scan note the presence of lesions, tumors,                          ☐ Yes                    ☐ Noor calcifications?For collection of aorta samplesDoes the CT scan note the presence of atherosclerotic disease            ☐ Yes                    ☐ Noor aneurysm?
TITLE: Cecret Workflow for SARS-CoV-2 Assembly and Lineage Classification 
AUTHORS: Erin L Young, Technical  Outreach and Assistance for States Team

[DESCRIPTION]
This protocol provides instructions to install and run the Cecret workflow as part of the StaPH-B Toolkit. Cecret produces SARS-CoV-2 consensus sequence assemblies from either single- or paired-end Illumina reads in fastq (or fastq.gz) format and assigns lineage classifications using Pangolin and Nextclade. This document applies to all whole-genome sequencing runs on the Illumina platform and downstream bioinformatics for public health laboratories.

For technical assistance, please contact: TOAST@cdc.gov

[STEPS]
SECTION: Software Dependencies
1. Load software dependencies
 

Within certain high-performance computing environments, these software can be loaded using GNU module commands similar to: 
 

SECTION: Installing StaPH-B Toolkit
2. The Cecret assembly workflow can be installed as part of the StaPH-B Toolkit using the following commands:
 

SECTION: Running Cecret Workflow
3. Run the Cecret workflow to perform SARS-CoV-2 genome sequence assembly and lineage classification
 

 

 

 

SECTION: Output Files
4. Information about output files
 

 

 

 


 


SECTION: Alternative Lineage Assignment
5. The SARS-CoV-2 consensus sequence assembly generated by the Cecret workflow can also be uploaded to other lineage assignment software.
SECTION: Alternative Lineage Assignment
5.1. Upload the consensus sequence for each sample to the Pangolin COVID-19 Lineage Assigner at:

https://pangolin.cog-uk.io/


Click the 'Start analysis' button:

  

The Pangolin COVID-19 Lineage Assigner returns the lineage classification and assignment probability:

  

SECTION: Alternative Lineage Assignment
5.2. Or upload the consensus sequence for each sample to the Nextclade clade assignment web portal at: 

https://clades.nextstrain.org/

  

The Nextclade server provides clade classification as well as QC metrics and a list of amino acid substitutions. A summary output file can be downloaded with the 'Export to CSV' button.