eamag/protocols-with-steps huggingface dataset:
TITLE: Primary Neuron Culture from Embryonic Rats
AUTHORS: Lucas Hampton, Paul Temkin

[GUIDELINES]
This protocol may also be used with P0 or neonatal mouse pups.

[STEPS]
SECTION: Procedure
2. Sacrifice time-pregnant rat (E18) by CO2 asphyxiation followed by secondary method (thoracotomy or cervical dislocation).

Note: All procedures are performed in compliance with AAALAC guidelines and are approved by the Biogen Institutional Animal Care and Use Committee.
SECTION: Procedure
3. Wash lower abdomen with 70% EtOH and make incision to reveal embryos.
SECTION: Procedure
4. One at a time, remove embryos from amniotic sac and decapitate. Cut below brain on rostral or caudal side and gently peel off skull. Remove brain and transfer to ice-cold HBSS. Repeat until desired amount of tissue is collected (~5x106 cells/brain). Decapitate any unused embryos.

Optional: If there are significant delays between collection and dissection, you may use ice-cold Hibernate EB Complete Media (BrainBits HEB500) to extend viability. Delays between collection and dissection will impact cell viability.
SECTION: Procedure
5. Transfer brains to 10cm petri dish with ~5ml ice-cold HBSS.
SECTION: Procedure
6. Separate hemispheres, remove meninges and olfactory bulb, and dissect out desired tissue. Immediately transfer tissue to ice-cold HBSS and store on ice until all tissue is dissected.
SECTION: Procedure
7. Decant or aspirate HBSS and add 10mL warm Dissociation Media (up to 10 brains/10mL). Rock tissue at 37oC from 30 minutes.
SECTION: Procedure
8. Allow tissue to settle and decant or aspirate Dissociation Media. Add 10mL warm Neutralization Media.
SECTION: Procedure
9. Remove all but 2mL of Neutralization Media, and gently triturate using P1000 pipette 10 times (avoid bubble formation). Let sit 2 minutes at RT.
SECTION: Procedure
10. Transfer supernatant (containing cell suspension) to new 15mL falcon tube, and add 2mL of Serum Media to the remaining settled tissue.
SECTION: Procedure
11. Repeat step 9, and then add the second cell suspension to the previously collected cells.
SECTION: Procedure
12. Spin down cell suspension at 30xg for 5 minutes, and then resuspend in 10mL of Serum Media. 
SECTION: Procedure
13. Assess cell count and viability, and plate as needed on poly-D-lysine coated cultureware with NB Media.
SECTION: Procedure
14. Perform 50% media change using NB media every 4-7 days depending on seeding density.
SECTION: Procedure
15. Optional: Add FUDR at DIV4 to inhibit mitotic cell growth.
SECTION: Procedure
1. Prepare Dissociation and Neutralization media fresh and cool 15mL HBSS (+Mg/Ca) on ice.
TITLE: FQ-LAMP Assay for Detection of CoV2 in Clinical Nasal Swabs
AUTHORS: Les Jones, Hemant K. Naikare, Yung-Yi C. Mosley, and Ralph A. Tripp

[DESCRIPTION]
COVID-19 is a public health challenge requiring rapid testing for detecting infections and transmission. Nucleic acid amplification tests (NAAT) targeting SARS-CoV-2 (CoV2) are used to detect CoV2 in clinical samples.Real-time reverse transcription-quantitative PCR (RT-qPCR) is the standard NAAT for CoV2, although Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP) is used in diagnostics. We show a sequence-specific RT-LAMP-based NAAT assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescent-quenched RT-LAMP assay (FQ-LAMP) using labeled primers and a quencher oligo. This assay can achieve rapid (30 min) and sensitive (1000 PFU/ml) fluorescent detection of CoV2 (WA1/2020), B.1.1.7 (Alpha), and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.

[STEPS]
SECTION: FQ-LAMP Sample Preparation
1. mix 20ul of clinical swab material with 20 ul of SPS buffer containing Proteinase K.
SECTION: FQ-LAMP Sample Preparation
2. Incubate at 37C for 15 minutes, then 95C for 5 minutes
SECTION: FQ-LAMP Sample Preparation
3. Sample is ready to use in FQ-LAMP at 2ul / 25ul reaction
SECTION: Prepare FQ-LAMP Master Mix
4. Determine the number of 25ul FQ-LAMP reactions needed and add 10%
SECTION: Prepare FQ-LAMP Master Mix
5. Mix the following in clean tube per 25ul FQ-LAMP reaction
SECTION: Prepare FQ-LAMP Master Mix
5.1. 2X WarmStart LAMP reagent 12.5ul
10X Detection Oligo/Primer Mix 2.5ul
deionized water 8ul
SECTION: Prepare FQ-LAMP Master Mix
6. Dispense 23ul of FQ-LAMP master mix to each required wells of a 96 well PCR plate
SECTION: Prepare FQ-LAMP Master Mix
7. Add 2ul of prepared sample in SPS buffer per assay
SECTION: Prepare FQ-LAMP Master Mix
8. Centrifuge the plate to settle all contents and seal the plate with appropriate cover material. 
SECTION: Run FQ-LAMP assay on real time PCR machine
9. Set up the real time machine to measure fluorescence in the FAM channel.
SECTION: Run FQ-LAMP assay on real time PCR machine
10. Set up the real time machine amplification program:
HOLD 65C / 30 minutes
HOLD 25C / 30 seconds
measure fluorescence
SECTION: Run FQ-LAMP assay on real time PCR machine
11. Record results
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background:</strong> The neutrophil-lymphocyte ratio (NLR) is increasingly known as an indicator of systemic inflammation. It is used as a predictor for clinical outcomes in cancers and cardiovascular disease. However, its relationship with intracerebral hemorrhage (ICH) is still disputed.</p>
<p><strong>Aims: </strong>We sought to evaluate the association between NLR and ICH in this meta-analysis.</p>
<p><strong>Methods:</strong> Potentially relevant articles were searched in PubMed, Cochrane Library, Medline and EMBASE until April 8, 2018, with English restriction. Efficacy outcomes included major disability at 90 days, short-term mortality or in-hospital mortality. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were pooled to estimate the prognostic role of NLR in ICH.</p>
<p><strong>Results</strong>:A total of 7 trials with 2176 patients were included. The results of our meta-analysis revealed that high NLR had a higher risk of major disability at 90 days (OR: 2.20; 95% CI: 1.27-3.81) and higher mortality at short-term (OR: 1.31; 95% CI: 1.02–1.68); without statistically significant association with in-hospital mortality (OR: 1.02; 95% CI: 0.91–1.15).</p>
<p><strong>Conclusions: </strong>Our meta-analysis proves that higher NLR was a predictor of major disability at 90 days and higher mortality at short term in patients with ICH, but not a predictor of in-hospital mortality.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Marine protists are important in all marine ecosystems, but many are not culturable and therefore difficult to study. The increasing development of single-cell techniques is allowing for more marine protists to be studied, but many single-cell protocols are only optimized for model organisms and do not perform well with marine plankton. The SMART-seq v4 manual specifically states that cells entering the pipeline should be washed and resuspended in cell culture-grade PBS, but most marine protists do not tolerate PBS. Additionally, many marine protists have mineral skeletons, the components of which may interfere with the chemistry of the SMART-seq kit. Calcium and Strontium both dissolve to form divalent cations which could saturate the kit's RNase inhibitor. When we synthesized cDNA directly from acantharians (SrSO<sub>4</sub> skeletons) without first extracting RNA, the cDNA was highly degraded with a 300 bp average fragment length. Therefore, it is desirable to perform RNA extractions from single marine protists before cDNA synthesis and amplification, but the amount of RNA in single marine protists is too low to be extracted with column-based kits (even the <a href="http://www.mn-net.com/Products/DNAandRNApurification/RNA/NucleoSpinRNAXS/tabid/10643/language/en-US/Default.aspx" target="_blank">MN Nucleospin RNA XS</a> kit suggested in the SMART-seq manual). To overcome these problems, we developed a column-free method for extracting RNA from single marine protists by making use of Agencourt RNAClean XP magnetic beads. When this protocol is followed, the synthesized cDNA is higher quality with a 2,000 bp average fragment length. </p>

[BEFORE_START]
<ul>
<li>Clean PCR-clean workstation and lab area with ethanol and RNase Zap. </li>
<li>Clean thermocyclers with RNase Zap. </li>
<li>Thaw reagents (except enzymes).</li>
<li>Allow reagents that need to be room temperature to incubate at room temperature for 30 min (RNAClean XP magnetic beads, AMPure XP magnetic beads, Bioanalyzer reagents)</li>
<li>Set thermocylcer programs and pre-heat thermocyclers.</li>
<li>Prepare fresh 80% ethanol.</li>
</ul>

[GUIDELINES]
<ul>
<li>Always wear clean RNase-free gloves. Clean gloves and work space with ethanol and RNase zap.</li>
<li>Perform single-cell RNA extractions and cDNA synthesis in a PCR-clean workstation, if possible. </li>
<li>It is good practice to have a separate set of pipettes for RNA and to use filter tips.</li>
<li>After cDNA synthesis, perform remaining steps in the general lab space. </li>
</ul>

[STEPS]
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TITLE: Enhanced convolutional neural network for plankton identification and enumeration
AUTHORS: Kaichang CHENG, Xuemin Cheng, Yuqi Wang, Hongsheng Bi, Mark C. Benfield

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an automatic plankton image recognition and enumeration system using an enhanced Convolutional Neural Network (CNN) and examined the performance of different network structures on automatic plankton image classification.</div></div>

[STEPS]
?. [The whole procedure]
The  whole procedure for the proposed method.This is a  flow chart illustrating the different steps and modules in the proposed automated plankton identification and enumeration procedure.
?. [Substeps in this procedure]
The several substeps for the proposed method.
?. [Substeps in this procedure]
Adaptive ROI extraction based on the following steps.Calculate the mean value of image intensity based on the following formula:Calculate the Mean Signal-to-Noise Ratio  value of the image  based on the following formula:Calculate the threshold of the image based on Sauvola's method:Binarize the image based on Sauvola's method.Extract ROIs based on the connected domain of the binarized image.
?. [Substeps in this procedure]
ROI enhancement based on the following method.A. Target feature enhancement based on the following formula:B. Background suppression based on the following formula:
?. [Substeps in this procedure]
Train CNN combined with multi-class SVM model based on the in situ plankton databset.The fully connected layers from the selected CNN models were used to describe sample features and were used as the input for the multi-class SVM model.  The multi-class SVM model is a one vs. one type,  and the linear relationship between the input and output can be described by the following formula:Save the trained model for next identification and enumeration.Note: you can get the dataset  at https://doi.org/10.6084/m9.figshare.8146283.
?. [Substeps in this procedure]
Identification and enumeration.The input of this procedure is the original in situ plankton image, and the output (statistical results of every plankton image for each class) is save in a file in the '. xlsx' format.Note: you can learn more details in our open codes in MATLAB language at https://github.com/KaichangCHENG/PIE-MC/tree/master/EnhancedCNN.
TITLE: Protocols for "The draft genome assembly of the critically endangered Nyssa yunnanensis, a plant species with extremely small populations endemic to Yunnan Province, China"
AUTHORS: Weixue Mu, Jinpu Wei, Ting Yang, Yannan Fan, Le Cheng, Jinlong Yang, Ranchang Mu, Jie Liu, Jianming Zhao, Weibang Sun, Xun Xu, Xin Liu, Radoje Drmanac, Huan Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Nyssa yunnanensis</span><span> is a deciduous tree species in the family Nyssaceae within the order Cornales. As only eight individual trees and two populations have been recorded in China’s Yunnan province, this species has been listed among China’s national Class I protection species since 1999 and also among 120 PSESP (Plant Species with Extremely Small Populations) in the </span><span style = "font-style:italic;">Implementation Plan of Rescuing and Conserving China’s Plant Species with Extremely Small Populations(PSESP)</span><span> (2011-2-15). </span></div><div class = "text-block"><span>We have assembled the draft genome assembly of </span><span style = "font-style:italic;">N. yunnanensis</span><span>. Using 10X Genomics linked-reads sequencing data, we carried out the </span><span style = "font-style:italic;">de novo</span><span> assembly and annotation analysis. The </span><span style = "font-style:italic;">N. yunnanensis</span><span> genome assembly is 1475 Mb in length, containing 288,519 scaffolds with a scaffold N50 length of 985.59 kb. Within the assembled genome, 799.51 Mb was identified as repetitive elements, accounting for 54.24% of the sequenced genome, and a total of 39,803 protein-coding genes were predicted.</span></div><div class = "text-block"><span>With the genomic characteristics of </span><span style = "font-style:italic;">N. yunnanensis </span><span>available, our study might facilitate future conservation biology studies to help protect this extremely threatened tree species. Protocols used include DNA-extraction, DNA Sequencing using the BGI-SEQ 500 (now known as DNBSEQ-G50) sequencer, and sequencing of 10X linked-reads.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Amalgam from various places.</p>

[BEFORE_START]
<p>This protcol is designed/re-optimized for staining 5e6 cells. However, sytoxgreen is supposed to be fairly robust to cell number changes, so variation from this should be tolerated (see Haase and Reed 2002). Remember, PBS messes with Sytoxgreen staining.</p>
<p> </p>
<p>Perform an <u><a href="https://wikis.nyu.edu/display/greshamlab/Ethanol+fix" target="_blank">Ethanol fix</a></u> in whatever condition you want. The following assumes that cells are be in 70% etOH and at -20C. You of course need to count the density of these fixed cells to proceed.</p>

[GUIDELINES]
<p>All spins are at 6000 rcf 1min RT. You will need 50mM sodium citrate buffer (~7.2 pH), a 5mM solution of sytoxgreen in DMSO, RNAseA (100mg/ml), and ProteinaseK (20mg/ml) solutions.</p>

[STEPS]
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TITLE: DNA/RNA Radiolabeling Protocol
AUTHORS: Liz O'Brien, Connor Tsuchida

[STEPS]
?. Incubate at  for .
37 °C
?. Prepare G25 columns (from GE, green box): vortex thoroughly, twist cap ¼ turn, snap off bottom, spin for  at  to get rid of liquid.
Centrifuge: 3000 33
?. Add  H2O to each labeling reaction after heat inactivation is done.
25 µl
?. Since 50 μl H2O were in bottom of tube and you add your 50 μl reaction, you should end with up to 100 μl of  labeled DNA/RNA.
?. Measure  of each reaction with the black rad counter on shelf to get cpm readings.
1 µl
?. Heat inactivate the PNK at  for .
65 °C
?. Apply entire reaction (now 50 μl total) to G25 column resin.
?. Spin for  at .
Centrifuge: 3000 33
?. Add  H2O to a labeled eppendorf tube, place G25 column in it.
50 µl
?. Set up labeling reaction:  AB1X μlDNA or RNA (10 pmoles)22.5 μl10x PNK buffer30.5 μlPNK enzyme41.5 μlP32-gamma-ATP5dH2O (DEPC for labeling RNA) to 25 μl
AB1X μlDNA or RNA (10 pmoles)22.5 μl10x PNK buffer30.5 μlPNK enzyme41.5 μlP32-gamma-ATP5dH2O (DEPC for labeling RNA) to 25 μl
Mix the DNA, buffer, enzyme, and H2O at the bench, and then add the DNA/enzyme mixture to ATP-filled tubes in a radioactive use area.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol may be used with electrocompetent cells prepared by you according to <a href="https://www.protocols.io/view/Making-your-own-electrocompetent-cells-imsv6m" target="_blank">this protocol.</a>

[BEFORE_START]
For control electroporation dilute pUC19 to 10 pg/&micro;l with Milli-Q water.

[GUIDELINES]
<strong>Appropriate Antibiotics for Your Application</strong><br /><br /><strong>Antibiotics for Plasmid selection</strong><br /><br /><table><tbody><tr><td>Antibiotic</td><td>Working Concentration</td></tr><tr><td>Ampicillin</td><td>100 &micro;g/ml</td></tr><tr><td>Carbenicillin</td><td>100 &micro;g/ml</td></tr><tr><td>Chloramphenicol</td><td>33 &micro;g/ml</td></tr><tr><td>Kanamycin</td><td>30 &micro;g/ml</td></tr><tr><td>Streptomycin</td><td>25 &micro;g/ml</td></tr><tr><td>Tetracycline</td><td>15 &micro;g/ml</td></tr></tbody></table><br /><strong>Electroporation Protocol</strong><br />The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/&micro;l with Milli-Q water.<br /><br /><strong>Calculation:</strong><br />If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)<br /><br />cfu/ &micro;g = (colonies counted*1000) / (0.00001 &micro;g pUC19)

[STEPS]
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TITLE: In vitro infection of PBMCs with dengue and Zika virus
AUTHORS: Yujao Zhao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details in vitro infection of human PBMCs with dengue/zika virus followed by multiparameter antibody labeling and data acquisition using CyTOF.The data using this protocol is available at ImmPort (immport.org) under study accession SDY1369.</div></div>

[STEPS]
TITLE: Expression and purification of Syp-VAMP2 complex into native nanodiscs
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This is a protocol for the purification of Syp-VAMP2 complex into native nanodiscs.

[STEPS]
SECTION: Mammalian expression constructs
1. Thaw Expi-HEK293 cells and passage 3 times
SECTION: Mammalian expression constructs
2. Using a T2A polycistronic vector containing the VAMP2 and Synaptophysin (Syp) genes with FLAG tag, express VAMP2 and Syp in Expi-HEK293 cells using ExpiFectamine transfection reagent for 2880 min hours.

SECTION: Mammalian expression constructs
3. Spin down transfected cells and rinse in ice-cold PBS.

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
4. Resuspend cells expressing VAMP2-Syp into lysis buffer (50 millimolar (mM) , 150 millimolar (mM) , 10 % volume ) supplemented with protease inhibitor tablets.

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
5. Lyse cells using nitrogen cavitation (600 psi, 15 min minutes).

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
6. Remove debris and nuclei using a 4000 rpm centrifugation spin for 10 min minutes.

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
7. Ultracentrifuge the clarified lysate at 200,000xg for 60 min hour at 4 °C to collect membranes.

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
8. Resuspend membranes in 1.5% polymer solution (ChloroSMA80) and incubate at 4 °C for 120 min hours.

SECTION: Solubilization of VAMP2-Syp into native nanodiscs
9. Post-solubilization centrifuge the sample at 200,000xg for 60 min  hour to remove insoluble material.

SECTION: Native nanodisc enrichment using affinity chromatography
10. Incubate native nanodiscs containing VAMP2-Syp complex with anti-FLAG resin   at 4 °C with rotation.

SECTION: Native nanodisc enrichment using affinity chromatography
11. Remove supernatant and wash beads extensively with 50 millimolar (mM) , 150 millimolar (mM) , 10 % volume. 

SECTION: Native nanodisc enrichment using affinity chromatography
12. Elute nanodiscs using buffer from Step 11 containing 3XFLAG peptide at 10 ug/ml concentration.
TITLE: LAMP assay
AUTHORS: Niels Appelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a protocol used to perform LAMP assays. It was adapted to </span><a href="https://international.neb.com/protocols/2016/08/15/warmstart-colorimetric-lamp-2x-master-mix-typical-lamp-protocol-m1800" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://international.neb.com/protocols/2016/08/15/warmstart-colorimetric-lamp-2x-master-mix-typical-lamp-protocol-m1800</span></a></div></div>

[STEPS]
?. Prepare a 10X primermix according to the following scheme:  AB1PrimerConcentration2FIP16 uM3BIP16 uM4F32 uM5B32 uM6LOOP F8 uM7LOOP B8 uM
AB1PrimerConcentration2FIP16 uM3BIP16 uM4F32 uM5B32 uM6LOOP F8 uM7LOOP B8 uM
?. Add the following components to a microcentrifuge tube  ABC1ComponentVolume added (25 uL  reaction)Volume added (10 uL  reaction)2Mastermix12.5  uL5  uL3Lamp  primers (10X)2.5  uL1  uL4Templatex  uLx  uL5MilliQ10  – x uL4 –  x uLAdd a no-template control to ensure template-specificity
ABC1ComponentVolume added (25 uL  reaction)Volume added (10 uL  reaction)2Mastermix12.5  uL5  uL3Lamp  primers (10X)2.5  uL1  uL4Templatex  uLx  uL5MilliQ10  – x uL4 –  x uL
?. Incubate at 65 C for 30 minutes.
?. Record the color of all samples. Yellowing of a sample indicates a positive result.
?. In case of doubt regarding the result of a sample, run it on an agarose gel. The presence of many bands, often not individually recognizable, indicates a positive result.
TITLE: Library Aligner
AUTHORS: ckremitz

[DESCRIPTION]
Protocol on how to run Library Aligner in FIVTools once sequencing data has been returned to us from the Sequencing Core.  It also has an option for checking the Representation of a gRNA library.

[STEPS]
1. Open FIVTools and click on the LA tab in the upper left section of the program.

 
2. Enter in your returned NGS link into the HTCF Link tab and the destination to download the files into Dest.

 
3. Press Download UZ to download the files and automatically unzip them.  This will deposit reads into your destination folder inside folders called FastQ and FastQ_gz.
4. Once downloaded, you can press the button Defaults under XML Settings: to return the left criteria back to the defaults.
5. In the left tab for PathReferenceList, enter in the pathway which contains the reference .txt file.  This file should contain the gRNA sequences that you wish to align to your reads.  If you wish to align to multiple different .txt files, just list the directory and not a specific file.
6. In the left tab for PathFastQToSearch, enter in the pathway to your FastQ files.

 
7. The rest of the settings can be adjusted to fit your schema. 
8. Press Start once you are ready to align your reads to your reference sequence list.
9. When LA is running, you will see several things happen on your screen.  You will want to be looking for the % of Unmapped Reads.  If this is 100%, then your reads are not matching anything in your reference list.  If you have a low % of Unmapped Reads, then that means they are hitting to reference reads, and that’s a good thing.
10. Once finished, LA will deposit a lot of files in your destination folder.  FastA files, ref_f.txt (what most people will want to look at - includes a counts table), ref_long.txt (fancier version used in Spotfire analysis), ref_TopSeq.txt (good for looking at your top hits if you get a low number of matches - answers the question: what are you matching to?).

 
11. If you wish to check the Representation of the gRNA libraries you’ve put into cells, you can do this at the bottom of Library Aligner.

 
12. Enter in the MOI you used to infect your cell line, the titer, and the Cell Factor.  Cell Factor is 1 if you were using hearty cells like U2OS or HeLa cells.  Cell Factor will be 2 if you were using delicate cells like iPSC cells. Select Merge P if you wish to merge SubPools together in the Representation Check, ie. MSPH1 through MSPH7 will be joined together.  Press Check Rep when ready.
13. You can also check your representation via the following website:  https://libalignerminiature20221013181154.azurewebsites.net/