eamag/protocols-with-steps huggingface dataset:
TITLE: Mitochondrial Respiratory Chain Complex Determination
AUTHORS: mitsjoecohen  

[DESCRIPTION]
Mitochondrial respiratory chain complex I (NADH oxidase) and mitochondrial respiratory chain complex II (succinate dehydrogenase) are the main elements of the electron transport chain (ETC). Complex I catalyzes the oxidation of NADH and complex II catalyzes the oxidation of succinic acid to fumaric acid. Subsequently, coenzyme Q forms coenzyme QH2, and finally leads to the decrease of terminal electron receptor O2. Mitochondrial respiratory chain complex III (cytochrome c oxidoreductase) is an essential protein for mitochondrial oxidative phosphorylation. Mitochondrial respiratory chain complex III is the gatekeeper of the mitochondrial respiratory chain and the main source of the third reactive oxygen species. Mitochondrial respiratory chain complex IV (cytochrome c oxidase) is the terminal electron receptor of the mitochondrial electron transport chain. Complex IV converts O2to H2O through the oxidation of cytochrome c, which is related to the synthesis of mitochondrial cell membrane ATP.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In this paper, we apply statistics and network analysis methods to explore the dynamic characteristics of three transaction networks. We download transaction data from the respective blockchain explorers. To the best of our knowledge, these are the largest datasets adopted in cryptocurrency analysis to date. We analyze the growth pattern of the accumulated network and find that unlike most networks, these cryptocurrency networks do not always densify over time. Then based on the datasets, we find that the monthly repetition ratios measured by either node or edge are relatively low. As such, studying the whole accumulated network, as done in most previous work, is not the appropriate way to understand the network dynamics. Hence we focus on coining the dynamics through computing the values of typical network measures on a monthly basis, and make a comparison among the three networks. </p>

[GUIDELINES]
<p>The program is written using Python 3.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Siamese fighting fish Betta splendens are notorious for their aggressiveness and accordingly have been widely used to study aggression. However, the lack of a reference genome has so far limited the understanding of the genetic basis of aggression in this species. Here we present the first reference genome assembly of the Siamese fighting fish.&lt;br&gt;We first sequenced and de novo assembled a 465.24 Mb genome for the B. splendens variety Giant, with a weighted average (N50) scaffold size of 949.03 Kb and an N50 contig size of 19.01 Kb, covering 99.93% of the estimated genome size. To obtain a chromosome-level genome assembly, we constructed one Hi-C library and sequenced 75.24 Gb reads using the BGISEQ-500 platform. We anchored approximately 93% of the scaffold sequences into 21 chromosomes and evaluated the quality of our assembly using the high contact frequency heatmap and BUSCO. We also performed comparative chromosome analyses between Oryzias latipes and B. splendens, revealing a chromosome conservation evolution in B. splendens. We predicted a total of 23,981 genes assisted by RNA-seq data generated from brain, liver, muscle and heart tissues of Giant, and annotated 15% repetitive sequences in the genome. Additionally, we resequenced other five B. splendens varieties and detected ~3.4M single-nucleotide variations (SNVs) and 27,305 indels.&lt;br&gt;We provide the first chromosome-level genome for the Siamese fighting fish. The genome will lay a valuable foundation for future research on  aggression in B. splendens.</p>

[STEPS]
TITLE: Proliferation assay
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
Assessing the impact of drugs and treatments on natural killer (NK) cell viability and expansion. 

[STEPS]
1. PBMCs or enriched NK cells (see Isolating NK cells from human blood products) are labeled with a permanent amine-reactive dye (CellTrace Violet Proliferation Kit, Cat. No:C34557, Thermo Fisher) according to the manufacturer's instructions. 
2. Cells are exposed to various experimental conditions, then harvested after 7 days of culture.
5. The samples are run on a flow cytometer (LSRII, BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., RRID:SCR_008520).

The proliferation of live (dead cell marker- and annexin V-) NK cells (CD56+/CD3-) and T cells (CD56-/CD3+) is assessed by dilution of the dye. 

Relative numbers of live NK cells and T cells for each treatment condition are obtained by running the flow cytometer at a constant flow rate and analyzing the number of each cell type obtained within a specified time limit e.g. 60s. 
3. Optional: If required, cells can be stained for apoptotic markers (FITC Annexin V, Cat. No: 556419, BD Biosciences) according to the manufacturer's instructions prior to antibody staining.
4. Cells are stained with a dead cell marker (LIVE/DEAD NEAR-IR, Cat. No: L34976, Thermo Fisher), PE-CY7 conjugated anti-CD56 (HCD56, BioLegend), and PE-CF594 conjugated anti-CD3 (UCHT1, BD BioSciences).
TITLE: Artificial Cerebrospinal Fluid VIII (ACSF.VIII)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to make Artificial Cerebrospinal Fluid VIII (ACSF.VIII).  ACSF.VIII is used for multiple applications including incubation of fresh human brain slices prior to electrophysiological recording.</div><div class = "text-block"><span style = "font-weight:bold;">Note: </span><span>Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: SEM materials checklist
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SEM materials checklist</div></div>

[STEPS]
?. [Bing from Fozo lab]
Poly-L-lysineSmall weight boats5 – 15mL conical tubesAnhydrous ethanol40mL 95% Ethanol25mL MethanolSterile WaterP1000 pipetteP200 pipetteP20 pipetteP200 compatible tipsP1000 compatible tips3–10mL serological pipet3 – 5mL serological pipetPipet aidSmall tweezersPermanent MarkerGlovesTimer
?. [Collect from JIAM]
Silica ChipsDouble-sided carbon tapeCPD –tiered chip holder
TITLE: BSCI:414--Lab 4 Plate Setup for COVID RT-qPCR
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this experiment is to show where saliva samples and positive and negative controls will be placed on a 96-well plate by an OpenTrons robot.</div></div>

[STEPS]
?. [Discuss Sample Types]
Review SalivaDirect for sample processing protocol: https://www.protocols.io/view/salivadirect-rna-extraction-free-sars-cov-2-diagno-bkjgkujw?step=11
?. [Inset Well Plate in Benchling Lab Notebook]
Open your Benchling Lab Notebook and insert "Lab 4" with heading "H1". Add pertinent links to protocols like the SalivaDirect protocol.
?. [Inset Well Plate in Benchling Lab Notebook]
We need to set up the format for our experiment in a 96-well plate. Add a 96-well well plate by selecting "Insert>Well plate>96-well plate".
?. [Inset Well Plate in Benchling Lab Notebook]
Add saliva samples 1 to 4, the no-template control and positive control using plasmid "pEX-A128-nCoV_CD". Make sure to "@-mention" the plasmid "pEX-A128-nCoV_CD" located in the BSCI:414 directory. Do not copy this to a new folder.
?. [Add qPCR Schedule to Lab Notebook]
Add a 2x7 table using "Insert>Table". Copy the qPCR schedule from Step 11 in SalivaDirect into the table . Use the values from the "NEB Luna" kit. Add a "Saliva Sample" row containing 5ul.Add a "TOTAL" row at the bottom and ensure reagents total 20ul.
TITLE: Immunoprecipitations and Western blots analysis
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Immunoprecipitations and Western blots analysis

[STEPS]
1. Immunoprecipitations were performed using
1-2 mg of total protein. Samples were incubated overnight with the indicated
antibody at 4° C, followed by the addition of 50 μl of protein G-agarose beads
(Thermo Scientific, Cat#20397). 



2. Immunoprecipitated proteins were recovered by centrifugation at 2,500× rpm for 2 min, washed three times with a buffer containing PBS and 0.15% Triton X-100. The resulting pellets were resuspended in 20 μl of 1X NuPAGE LDS sample buffer (Thermo Scientific #NP007) and
incubated at 95 °C for 10 min. Samples were separated by NuPAGE 4 to 12%
Bis-Tris polyacrylamide gels (Thermo Scientific #NP0335BOX), and transferred to
a 0.2 µM PVDF membrane (Thermo Scientific #LC2002), using the Mini Blot Module
system (Thermo Scientific). 
3. PVDF membranes were first fixed with 0.2% PFA 1x PBS per 30 min at room temperature. Then, membranes were washed three times for 10 min in PBS with 0.1% Tween 20 Detergent (TBST) and blocked for 1 h in TBST buffer containing 5% dry milk, and then incubated with the indicated primary
antibody for 1 h in blocking solution, washed three times for 10 min each and
incubated with HRP-conjugated secondary antibodies (RRID:AB_2819160),
(RRID:AB_2755049). 


4. After antibody incubations, membranes were again washed three times with TTBS buffer, and protein bands were visualized using the ChemiDoc Imaging System (BioRad) and quantified with Image Lab software version 6.1 from BioRad (RRID:SCR_014210)
http://www.bio-rad.com/en-us/sku/1709690-image-lab-software.
TITLE: Annotation for Fungi
AUTHORS: Sebastian Bassi, Claudia V. Gonzalez, Tristan Yang

[DESCRIPTION]
Protocol to annotate a fungi genome

[STEPS]
SECTION: Setup
1. Install Docker

If you don't have Docker already, install it. There are two versions, Docker Engine (also known as CE) and Docker Desktop. The Desktop version is more user friendly but since may require commercial license for large enterprise, this tutorial is based on the Docker engine. Both version will work in this protocol. Linux users can install both Docker CE and Desktop, while macOS and Windows users should install Docker Desktop.

Follow the installation instructions from https://docs.docker.com/engine/install/ 


SECTION: Run sspace_longread
3. Run the following command (replace /your_dir for the base directory where you have your data



 

SECTION: Run BWA Index
5. Run the following command (replace /your_dir for the base directory where you have your data


 


SECTION: Setup
2. Get your data

You will need fastq data (long read, called ID.fastq), short reads, and the assembly data. 
SECTION: Run Gapcloser
4. Run the following command (replace /your_dir for the base directory where you have your data


 


SECTION: Run fastp
6. Run the following command (replace /your_dir for the base directory where you have your data)


 

SECTION: Run BWA mem
7. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 

SECTION: Run SAMTOOLS
8. SAMTOOLS View, Sort and Index

Run the following command (replace /your_dir for the base directory where you have your data).


 



SECTION: Pilon
9. Run the following command (replace /your_dir for the base directory where you have your data).


 

SECTION: Funannotate
10. 
Funannotate Clean and Sort

Run the following command (replace /your_dir for the base directory where you have your data).

 

SECTION: RepeatMasker
11. 
Run the following command (replace /your_dir for the base directory where you have your data).


 


SECTION: Fuannotate Predict
12. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 

SECTION: Interproscan
13. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 


SECTION: Funannoate annotate
14. Run the following command (replace /your_dir for the base directory where you have your data)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>CZV8-proto agar </strong></p>
<p>                                         <strong><u>500 mL</u></strong>                                        <strong><u>300 mL</u></strong></p>
<p>Czapek-Dox broth              22.7 g                                         13.62 g</p>
<p>agar                                   5 g                                              3 g</p>
<p>sorbitol                              91.1 g                                         54.7 g                       </p>
<p>centrifuged V8 juice         100 mL                                        60 mL</p>
<p>add H<sub>2</sub>O to                       500 mL                                       300 mL</p>
<p>adjust to pH 6.0 with NaOH</p>
<p>autoclave and pour thin plates (approx. 10mL)</p>

[STEPS]
?.
TITLE: The efficacy and safety of preoperative glucocorticoid in herniorrhaphy: A systematic review and meta-analysis (protocol)
AUTHORS: Jun Watanabe, Kazuma Rifu, Takehiro Kagaya, Kazuhiko Kotani, Naohiro Sata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Introduction</span><span>: Postoperative pain is one of the main reasons for delayed recovery after herniorrhaphy. Preoperative glucocorticoid administration may lead to improved recovery after surgery. The present study will aim to examine the efficacy of preoperative glucocorticoids on recovery after herniorrhaphy.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods</span><span>: Random controlled trials (RCTs) examining the efficacy of preoperative glucocorticoids after herniorrhaphy will be searched using the electronic databases and the trial registries. Meta-analyses will be performed using random-effects models. The Grading of Recommendations, Assessment, Development, and Evaluation approach will be used to assess the certainty of evidence.</span></div></div>

[STEPS]
TITLE: Bioflux Analyses
AUTHORS: Tobias Weise, Bettina Boettcher, Slavena Vylkova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Biofilm formation under shear flow conditions was monitored using the Bioflux1000 device (Fluxion Biosciences, Inc.). In short, Candida albicans overnight cultures were washed in pre-warmed RPMI medium. Cells were seeded for 2-5 sec from the outlet well into the channels of Bioflux1000 flow chambers, which were primed before with warm medium. The cells were allowed to adhere to the channels for 90 min without any flow, followed by removal of non-adherent cells by flowing fresh, pre-warmed RPMI medium for 5 sec. Shear flow was set for time series experiments over 24 h biofilm formation and images were captured every 20 min. Two channels were investigated in parallel having a 10 × magnification to allow a direct comparison between a mutant and a reference (wild type) strain. Image capturing and stacks to movies was performed using the MetaMorph® Software (Molecular Devices).</div><div class = "text-block">Source material provided as AVI files was converted into single TIFF images as well as data frames containing meta data annotations. The individual image contains two growth chambers (wild type and mutant) separated by four edge lines. Images were rotated automatically to vertical alignment in order to carry out an automated chamber detection and analysis. The mean pixel intensity (i. e. grey scale value; reflecting cell density) of the individual chamber was calculated and added into the respective data frame. </div><div class = "text-block"><span>An ODE model reflecting the logistic growth as well as the lag phase was fitted to the individual experiments. Fitting was carried out by minimising a cost function (unweighted least-squares-based) using the </span><span style = "font-style:italic;">Nelder-Mead </span><span>algorithm. Growth rate time series generated from the fitted model were used to compare wild type and mutant regarding the maximum observed growth rates at their respective time points.</span></div><div class = "text-block">All computations were performed using the programming language python (version 3.6.9) and the additional packages numpy (version 1.16.2), opencv-python (version 4.1.1.26), pandas (version 0.25.0), scipy (version 1.3.1) and scikit-image (version 0.15.0).</div></div>

[STEPS]
TITLE: Adrenal Chromaffin Cell Cultures
AUTHORS: Ellen Kantar, David Sulzer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about culturing of adrenal chromaffin cells from rats.</div><div class = "text-block">For rat-derived cultures, adrenal glands from 7- to 12-day-old Sprague Dawley rats are dissected in ice-cold Hanks Balanced Salt Solution (HBSS).</div></div>

[STEPS]
?. Animals are decapitated (anaesthetize the animals with Ketaset® KETAMINE, FORT DODGE® #NDC-0856-2013-01 if decapitation is not an approved protocol).
?. Decapitate and pin the body belly-down. Spray with .
[ethanol]
?. Cut the skin along the spinal column (it’s easier starting from the neck) and pull it out to both sides, using a scissor to separate the skin from underlying tissue. The back of the body is now open.
?. Grab the vertical column with forceps and pull it up while cutting along both sides through the ribs. It’s better to cut both sides simultaneously (alternating cuts on each side as you work back). Eventually you will see the diaphragm. At this point, open the scissors and place onto the diaphragm approximately 1/3 of the way up from the bottom. Pull the spine up while holding the diaphragm down. The abdominal cavity should tear open exposing, among other organs, the two kidneys with the adrenal glands on top. These should be readily visible.
?. Remove the glands with fine forceps (curved are the best, pinch off the tissue under the glands and pull up) and put them into ice-cold HBSS (Ca2+/Mg2+ free).
?. Adrenal glands are encased by adipose tissue and a capsule. Remove the capsule (using two fine forceps, try to pull the capsule open like a bag of potato chips, then holding the capsule with one tweezer, use the other to “roll” off the gland). Cut the adrenal glands in half (or thirds depending on size of glands).
?. After several washes with HBSS (use a sterile plastic transfer pipette), the tissue is digested with collagenase IA solution in , for about - at  with stirring. Stop the digestion as soon as the solution starts to turn cloudy.
[HBSS , Ca2+/Mg2+-free, (250-350 U/ml, Worthington)]
37 °C
?. The digested tissue is rinsed with HBSS and triturated gently in a solution containing  and . For trituration use large bore tech-tips, and if needed, medium bore tech-tips.
[heat inactivated fetal bovine serum]
[DNase I]
Please refer to our “Ventral Midbrain Cultures” protocol for the instruction on how to prepare the tech-tips.
?. Dissociated cells are centrifuged at  to form a pellet and then resuspended in a culture medium comprised of DMEM, , , , and  .
Centrifuge: 1000 34, 3 min
[fetal bovine serum]
[penicillin]
[streptomycin]
[Glutamine]
?. The cell suspension is plated onto poly-D-lysine and laminin-coated glass wells in 50 mm dishes (cells from 5 rat 10-day-old pups onto 16 dishes) and, after , the dishes are flooded with the culture medium ().
[per dish]
Please refer to our “Ventral Midbrain Cultures” protocol for the instruction on how to prepare and coat the dishes .
?. Cells are maintained in a 5% CO2 incubator at .
37 °C
All measurements are conducted between 1 and 7 post-plating days.
TITLE: Fear Conditioning
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
Behavioral assay to measure fear learning and memory in mice.

[STEPS]
SECTION: Fear Acquisition
1. Habituate mice to the room. Set up fear conditioning chambers as well as protocols (listed below) in VideoFreeze software.
2. Place individual mice in a lit fear conditioning apparatus scented with peppermint spray.
3. Protocol:
2 min baseline followed by 5 tone-shock pairings where the shock follows the tone
shock = 0.5mA, 1 s is delivered after the end of the tone (75dB, 2kHz, 19 s)
empty trace intervals between the shocks last 1 min
SECTION: Contextual Test
6. Goal: Measure the subjects' fear response to the environment where fear conditioning was received
4. After the protocol runs for each mouse, place the mouse back in the home cage and clean the fear conditioning chamber thoroughly.
SECTION: Contextual Test
7. 24 hours after fear acquisition: Mice are placed in a lightened FC apparatus scented with peppermint spray.
SECTION: Cued Test
9. Goal: Measure the subjects fear response to the CS, and ideally no other features of
the training environment. The key is to alter the chamber so the subjects are exposed to a new set of environmental cues. 
SECTION: Primary Analyses
12. Fear conditioning acquisition 
% Component freezing for baseline, each tone (if used), shock, and interstimulus interval 
Graphs for each component should be graphed separately
Average motion
Context
% Component freezing per minute and total 
Per minute and total will be on separate graphs
Average motion per minute and total
Cued
%Component freezing for baseline, each tone, and interstimulus interval
Graphs for each component should be graphed separately
Average motion for baseline, each tone, and interstimulus interval
5. Contextual test is performed 1440 min after the fear acquisition test. Cued Test is performed at least 120 minafter the contextual test. 

SECTION: Cued Test
10. Mice are placed in a lightened FC apparatus which is altered by placing a smooth floor insert on shock grid and black triangles on top.

Limonene spray is used, instead of peppermint scent. 
SECTION: Contextual Test
8. Protocol: Neither cue nor shock are delivered during this test session (Duration 450 s)

SECTION: Cued Test
11. Protocol: 
Baseline: 2 min followed by five tone-cues
No shock is delivered after the end of the tone (75dB, 2 kHz, 19 s)
TITLE: Donor case selection protocol
AUTHORS: kyu sang han, Sashank Reddy, peihsun.wu, Denis Wirtz

[DESCRIPTION]
Inclusion
Age 18-35 or 50 and over
English Speaking
Able to provide consent
Undergoing reconstructive or aesthetic procedures necessitating skin excision at the scalp or abdomen
 
Exclusion
 
Known collagen disorder
Strong personal history of keloid formation
Deemed unfit for general anesthesia
Active infection of skin where samples may be taken

[STEPS]
SECTION: Sample collection guideline
1. Patient samples will be obtained from skin that is to be discarded from patients presenting for reconstructive or aesthetic procedures necessitating skin excision. No other procedures or follow-up is needed for research purposes.
SECTION: Sample collection guideline
2. Inclusion/Exclusion Criteria

Inclusion
Age 18-35 or 50 and over
English Speaking
Able to provide consent
Undergoing reconstructive or aesthetic procedures necessitating skin excision at the scalp or abdomen
 
Exclusion
 
Known collagen disorder
Strong personal history of keloid formation
Deemed unfit for general anesthesia
Active infection of skin where samples may be taken
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How to make a IPTG &ndash; 1 M (100 x) Stock Solution

[GUIDELINES]
Make a <a href="http://store.p212121.com/IPTG/" target="_blank">IPTG</a> &ndash; 1 M (100 x) Stock Solution

[STEPS]
?. 
?. 
?. 
?.