eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Materials:</strong><br /><br /></p>
<p>1) 60-65°C heat block or water bath</p>
<p>2) Microfuge</p>
<p>3) 1.5 and 2.0 mL microfuge tubes (screw-cap)</p>
<p>4) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl<sub>2</sub></p>
<p>5) Triton X-100<br />6) DNAse I, 2.0 mg/mL in 50 mM Tris-HCl, pH 8.0. Store in 110 µL aliquots at -20°C  DO NOT REFREEZE UNUSED MATERIAL, DISCARD. </p>
<p>7) Proteinase K, 2.0 mg/mL in 50 mM Tris-HCl, pH 8.0.  Autodigest for 60 min at 37<sup>o</sup>C before use. Store in 1.0 mL aliquots at -20<sup>o</sup>C. Can be refrozen unless the material has fallen out of solution. </p>
<p>8) 10% Na sarcosyl</p>
<p>9) CHCl<sub>3</sub>:Isoamyl alcohol (24:1)</p>
<p>10) 500 mM EDTA, pH 8.0</p>
<p>11) 3 M NaOAc</p>
<p>12) 100% EtOH</p>
<p>13) Buffer-saturated phenol</p>
<ul>
<li>Preparation: Thaw 100 gm bottles of phenol at 60-65<sup>o</sup>C. Add a stir bar, 100 mL of 500 mM Tris-HCl, pH 8.0 and 0.1 gm 8-hydroxyquinoline. Stir and allow the phases to separate at 4<sup>o</sup>C overnight. Aspirate off the upper aqueous layer and add 75 ml of 100 mM Tris-HCl, pH 8.0, 0.2% 2-mercaptoethanol (2-ME) and stir. Allow the phases to separate at 4<sup>o</sup>C for several hours to overnight and remove the upper aqueous layer. Repeat the 75 mL addition 2X, leaving the final phase on the phenol. Store at 4<sup>o</sup>C. </li>
</ul>
<p>14) 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (1X TE)</p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Galvanic vestibular stimulation (GVS) is a method that stimulates the vestibular afferents with a small current, triggering a motor reflex response in the posture-controlling muscles. This response can be captured by electromyography (EMG) and is called vestibular evoked myogenic potential (VEMP). The VEMP triggered by GVS (galvanic-VEMP) assesses the central pathways of the vestibular system. When the EMG responses are recorded in the lower limbs, galvanic-VEMP provides information about the function of the vestibulospinal tract extending from the cervical to the lumbar spine. Galvanic-VEMP is a safe, low-cost, and easily performed test which has been used to investigate spinal cord function in cases of trauma, tumor, ischemia, and infection. Our research group has been using galvanic-VEMP to follow patients with diseases that cause postural disabilities, such as Human T-cell lymphotropic virus type 1-associated myelopathy (HAM), Schistosomal myeloradiculopathy, and Parkinson’s disease. This protocol aims at showing how to perform VEMP triggered by galvanic vestibular stimulation with the EMG responses being recorded in the lower limbs.</p>

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TITLE: Self-perception of Physical Appearance of Adolescents and Associated Factors in Addis Ababa, Ethiopia
AUTHORS: ziyadahm, ziyadahm, Semira Ahmed2, Aynye Woldekiros Negese

[DESCRIPTION]
Introduction: Establishing a positive body image is a critical factor for adolescents’ physical and mental health, as it determine self-confidence, sustainable individual growth and development throughout their lives. This reality is need to be supported with evidences generated locally. However, there is a lack of information in this regard in the study setting. Thus, this study aimed to close this evidence gap by assessing self-perception of physical appearance and its associated factors among adolescents in Addis Ababa.

Method and material: A community-based cross-sectional study design was used to conduct the study. At the end of multistage sampling procedure, a systematic random sampling techniques was employed to select 308 study participants from selected districts. The data collectors were trained before the data collection and supervised during the data collection period, and the questionnaire was pretested. Logistic regression was employed to identify the predictor variables. Statistical significance was considered at P <0.05 with adjusted odds ratios calculated at 95% CIs.

Result: The overall good self-perception of one’s physical appearance was 48.4% [95% CI=48.8, 541]. Having BMI between 18.5kg/m2-24.9kg/m2(AOR=2.56; 95% CI: 1.45, 4.54), presence of enough sport fields in the school(AOR=1.89; 95% CI: 1.09, 3.29), having daily access to internet services (AOR=1.69; 95% CI: 1.07, 2.94), following Ethiopian movies/cinemas (AOR=2.46; 95% CI: 1.46, 4.15), and regularly following Western movies/cinemas (AOR=2.0; 95% CI: 1.11, 3.59) were significantly associated with self-perception of one’s physical appearance. 

Conclusion: According to this study, good self-perception of one’s physical appearance was low. Adolescents and their family need to work to bring normal body weight and height, which in turn brings their normal body mass index, which will increase good self-perceptions of their physical appearance. Schools need to increase access to enough sport fields for students to strengthen their physical fitness, which increases their good self-perception of their physical appearance. 

Key Words: Self-perception, Adolescent, Physical Appearance, Addis Ababa, Ethiopia

[STEPS]
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TITLE: Quantitative analyses of the ultrastructural features of dopaminergic axon terminals
AUTHORS: Natalie M Doig, Peter J Magill

[DESCRIPTION]
The release of dopamine from axons is critical for normative brain function and behaviour. Impaired or otherwise inappropriate dopamine release often correlates with changes in the ultrastructure of dopamine neuron axons that can assessed with electron microscopy. Here, we provide two protocols that can be used serially to, first, help the user process animal brain tissue for electron microscopy and, secondly, help the user undertake quantitative analyses of the ultrastructural features of dopaminergic axon terminals in the brain. 

Protocol #1 describes how to prepare brain tissue, carry out pre-embedding immunohistochemistry for tyrosine hydroxylase as a marker of dopaminergic axons, and then make tissue sections ready for electron microscope. 

Protocol #2 details how to examine and image ultrathin sections of tissue using a transmission electron microscope and then how to analyse the digital images.

[STEPS]
TITLE: Expression and purification protocol of GST-TAX1BP1
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the expression and purification protocol of GST-TAX1BP1.</div></div>

[STEPS]
?. [Protein expression]
Transform the E.Coli BL21DE3 cells with a plasmid encoding for GST-TAX1BP1 and plate them on Amp plate.
?. [Protein expression]
Carry out protein expression in  LB medium, induce with  IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at .
1.5 L
18 °C
?. [Protein expression]
Harvest the cells by spinning at  for  at  and stock at  until purification.
Centrifuge: 4500 34
4 °C
-80 °C
?. [Protein purification]
Follow the GST batch purification by Size Exclusion Chromatography.
?. [Protein purification]
Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at  at  for
Centrifuge: 16000 33
4 °C
?. [Protein purification]
Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at  with gentle shaking for , apply to a gravity column, and wash extensively with Wash Buffer.
4 °C
?. [Protein purification]
Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer at .
4 °C
?. [Protein purification]
Pool the peak fractions containing pure protein, snap-frozen in liquid nitrogen and store at .
-80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The present work provides information about the distribution, quantity and nature of lipophilic substances beneath the surface in air- and kiln-dried Scots pine (<em>Pinus sylvestris</em> L.) sapwood boards. Samples were taken from knot-free sapwood surfaces and the composition of lipophilic extractives, phenols and low-molecular fatty/resin acids layers at different nominal depths below the surface was studied gravimetrically, by UV-spectrometry and by gas chromatography-mass spectrometry (GC-MS).</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was developed in order to remove secondary compounds which inhibited restriction enzyme digestion of <em>Xanthorrhoea</em> genomic DNA. It has also been used to successfully obtain high quality, high weight DNA from other recalcitrant plant tissues, including <em>Eucalyptus</em>, <em>Eremophila</em>, <em>Spyridium</em>, and various Malvaceae, Rutaceae, Orchidaceae, and Fabaceae species.</p>
<p> </p>
<p>It is based on a modified CTAB protocol from Doyle and Doyle (1990), and includes modifications from Shepherd and McLay (2011) and Tibbits <em>et al</em> (2006).</p>

[BEFORE_START]
<p>Prepare the following reagents:</p>
<p> </p>
<p>Tris-HCl pH8 (1M)</p>
<p>EDTA (0.5M)</p>
<p>NaCl (5M)</p>
<p>STE</p>
<p>CTAB </p>
<p>NaCl:BSA 5:1</p>
<p>Sodium acetate (2.4M)</p>
<p>Tris-HCl pH8 (10mM)</p>
<p>70% ethanol</p>
<p> </p>

[GUIDELINES]
<p>This protocol requires standard molecular laboratory equipment and chemicals. There are three solution additives which were critical for removing inhibiting secondary compounds from <em>Xanthorrhoea</em> tissue; STE, NaCl:BSA, and NaAC. It is possible that not all these additives are required for all plant tissues. However, all three are used in our lab to obtain very clean DNA from a variety of families. </p>
<p> </p>
<p>It is possible to include the NaAC wash step with the isopropanol precipitation, but performing it at the end seems to produce better quality DNA. </p>

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Culley Protocol (from Culley &amp; Steward (2007), <a href="http://aem.asm.org/content/73/18/5937.full" target="_blank">New genera of RNA viruses in subtropical seawater, inferred from polymerase gene sequences</a>, Applied &amp; Environmental Microbiology 73(18):5937-5944).

[GUIDELINES]
<strong>Supplies needed (per 100 mL sample):&nbsp;&nbsp;&nbsp;&nbsp;</strong><br />&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />Sample (0.2 &micro;m filtered seawater)One 60 mL syringe<br /> One Anotop (0.02 &micro;m filter)<br /> One piece of parafilm<br /> One piece of aluminum foilOne Ziploc freezer bag<br /> Sharpie (ultra-fine)<br /> Can crusher

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TITLE: Spinfection for Suspension and Adherent Cells
AUTHORS: Kevin Wilkins

[DESCRIPTION]
This protocol is for infection of adherent cells (such as HEK293t) with prepackaged lentivirus. 
Different cell lines will have different infection rates, and the infectivity of lentivirus stocks will also depend and vary. Optimization will be required to obtain infection, as well as to determine the appropriate proportion of cells and virus to obtained the desired MOI>

[BEFORE_START]
Make and prepackage your virus.

[GUIDELINES]
When using lentivirus, adhere to the proper safety measures and precautions. Proper care should be taken that all waste is disposed in biohazard containers or bleached. Double gloves are reccomended.

[STEPS]
SECTION: Plating cells
1. For HEK293t cells, plate 50,000 cells/well in 0.5 mL of DMEM on 24-well plate the day prior.
SECTION: Spinfection
2. 30+ min before spinfection, make sure the table-top centrifuge is at 30 °C .

SECTION: Spinfection
3. Add lentivirus and polybrene to each well.
SECTION: Spinfection
3.1. Virus amount
Virus titer will vary, so it is useful to add different amounts of virus to well to determine the appropriate titer. For a well of a 24-well plate, virus amounts can range from 5 μL to 500 μL.
SECTION: Spinfection
3.2. Polybrene amount
For most cell types polybrene increases infection efficiency and should be used at a final concentration of 8 μg/mL. Example dilution: add 0.8 uL (160 μg) polybrene stock (stock at 100mg/mL) to 0.5 mL media.
SECTION: Spinfection
4. Spin at 1000 rcf for 2 hrs at 30 C. 
1000 rcf, 30 °C 

SECTION: Spinfection
4.1. It is OK if the temperature starts off between 20-30C.
SECTION: Spinfection
5. Remove plate from centrifuge and place in hood. Douse with ethanol and wait 10 minutes. Place in incubator.
SECTION: Spinfection
6. Infection will be achieved within 2-3 days.
TITLE: Protocol for sampling and transport of nose- and thoratsamples in the Fit Futures study
AUTHORS: Dina Stensen, Anne-Sofie Furberg, Karina Olsen, Gunnar Skov Simonsen, Johanna UE Sollid, Lars Småbrekke, Guri Grimnes, Cristopher Sievert Nielsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The aim of the study is to evaluate prevalence of </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> in a youth population. This samples will be connected to other possible riskfactors for </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> carriage. </span></div></div>

[STEPS]
?. [Nasal samples]
Moisture the rayon-tipped swab in 2-3 drops of sterile NaCl 0,9 mg/ml. There should be two samples from the nasal cavity.
?. [Nasal samples]
Lead the rayon-tipped swab carefully into the nasal cavity approximately 1-2 cm and rotate with delicate pressure 3 times in both nasal cavity. The same swab should be used for both anterior nares.
?. [Nasal samples]
Place both swabs in the Coban transport medium.
?. [Nasal samples]
Label the transport medium with testnumber and nasal swab 1 and 2. Nasal swab 1 is used for detection of bacterial analysis, and swab 2 for microbiome analysis.
?. [Throat samples]
Moisture the rayon-tipped swab in 2-3 drops of sterile NaCl 0,9 mg/ml. There should be two samples from the throat.
?. [Throat samples]
Ask the participant to open his/her mouth. Press the tongue spatula against the tongue and ask the participant to say "aaa". This will simplify the sampling because this will better the view of the tonsil region.
?. [Throat samples]
Lead the rayon-tipped swab into the oral cavity and swab with moderate pressure on both tonsil regions (palatine tonsils). If the participants has had a former tonsillectomy/tonsillotomy the swab should be taken from the area of the removed tonsils. Use the same swab on both tonsils.
?. [Throat samples]
Place both swabs in the Coban transport medium.
?. [Throat samples]
Label the transport medium with testnumber and throat swab 1 and 2. Throat swab 1 is used for detection of bacterial analysis, and swab 2 for microbiome analysis.
?. [Throat samples]
Wash hands with soap and water or use hand disinfection after procedure
?. [Nasal samples]
Wash hands with soap and water or use hand disinfection after procedure
?. [Transport]
The swabs should be placed in a suitable rack. If not picked up immediately, the samples should be placed in refrigerator awating pickup.
TITLE: UMN-Mouse-rnaSeq_skeletal_muscle
AUTHORS: Xu Zhang

[DESCRIPTION]
Protocol for mouse rnaSeq for skeletal muscle

[STEPS]
1. Description Source name	Skeletal muscle Organism	Mus musculus Characteristics	stain: C57BL/6 age: 6 month Treatment protocol	NA Growth protocol	NA Extracted molecule	total RNA
2. Process Extraction protocol	Quadriceps muscle were collected in Trizol solution (Invitrogen, Carlsbad, CA, USA) and RNA isolation was performed following the instructions. cDNA libraries were prepared according to the manufacturer’s instructions for the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA).  Library strategy	RNA-Seq Library source	transcriptomic Library selection	cDNA Instrument model	Illumina HiSeq 4000
3. Data processing Base-calling was performed using Illumina's RTA version 2.7.7. Raw RNA sequencing paired-end reads were processed through the Mayo RNA-Seq bioinformatics pipeline, MAP-RSeq version 3.1.3. In this process, the fast, accurate and splice-aware aligner, STAR (v2.6.1c), was used to align reads to the reference mouse genome build mm10. Subsequently, gene and exon expression quantification was performed using the Subread featureCounts (v1.5.1) [3] package to obtain both raw and normalized (FPKM - Fragments Per Kilobase per Million mapped) reads. Genome_build: mm10 Supplementary_files_format_and_content: a table of raw counts of all samples
4.
TITLE: LRRK2 cloning, plasmid construction, and mutagenesis
AUTHORS: David Snead, Yu Xuan Lin

[DESCRIPTION]
Protocol for Cloning, plasmid construction, and mutagenesis of LRRK2 and LRRK2-RCKW as done by Leschziner and Reck-Peterson Labs.


Original protocol by David Snead  and Yu Xuan Lin.

[STEPS]
SECTION: Cloning, plasmid construction, and mutagenesis
1. The DNA coding for LRRK2-RCKW residues 1327 to 2527 (taken from Mammalian Gene Collection) was PCR-amplified using the forward primer TACTTCCAATCCATGAAAA491AGGCTGTGCCTTATAACCGA and the reverse primer TATCCACCTTTACTGTCACTCAACAGATGTTCGTCTCATTTTTTCA. 
The DNA coding for LRRK2 was codon-optimized for Spodoptera frugipera (Sf9) cells and synthesized by Epoch Life Science.
SECTION: Cloning, plasmid construction, and mutagenesis
2. The DNA for either LRRK2 and LRRK2-RCKW, containing a N-terminal His6-Z-tag and TEV protease cleavage site, was cloned into a pFB-6HZB vector (SGC) by ligation-independent cloning, RRID:Addgene_53641 
SECTION: Cloning, plasmid construction, and mutagenesis
3. LRRK2 variants were generated using Q5 Site-Directed Mutagenesis Kit (NEB).
SECTION: Cloning, plasmid construction, and mutagenesis
3.1. Primers for G2019S mutant:
Forward: gccaagatcgctgactacagcattgcccagtactgttgc
Reversed: gcaacagtactgggcaatgctgtagtcagcgatcttggc

Primers for I2020T mutant:  
Forward: ccaagatcgctgactacggaactgcccagtact
Reversed: agtactgggcagttccgtagtcagcgatcttgg 

Using the following thermocycler conditions.
98 °C 30 s 
( 98 °C 10 s 
  50-72 °C 30 s 
  72 °C 13 min 
  72 °C 2 min ) X25 cycles
4 °C Hold.
SECTION: Cloning, plasmid construction, and mutagenesis
4. PCR products were subject to a KLD reaction (NEB), followed by a transformation into DH5α competent cells and plated on LB with antibiotics.
SECTION: Cloning, plasmid construction, and mutagenesis
5. Colonies were picked and grown overnight for DNA extraction using Qiagen miniprep kit.
SECTION: Cloning, plasmid construction, and mutagenesis
6. Extracted DNA plasmids were submitted for sequencing. 
The resulting plasmids were utilized for the generation of recombinant Baculoviruses according to the Bac-to-Bac expression system protocol (Invitrogen).
TITLE: Sample preparation for Illumina MiSeq Dual Index metabarcoding
AUTHORS: Xiaohuan Sun, Yuehua  Hu, Zewei  Song

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol shows amplicon sequencing samples preoared by Illumina Dual Index strategy, and sequened with Illumina MiSeq (RRID:SCR_016379) for bacteria and microeukaryotes.</div></div>

[STEPS]
?. Perform the first PCR (duplicates of each sample) using Illumina adaptor attached primers that target the gene of your choice. Here we present the protocol using the eukaryotic primers 5.8S and ITS4.  AB1The primer sequences25.8SR_NexteraTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3TCGATGAAGAACGCAGCG4ITS4_NexteraGTCTCGTGGCTCGGAGATGTGTATAAGAGACAG5TCCTCCGCTTATTGATATGC
AB1The primer sequences25.8SR_NexteraTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3TCGATGAAGAACGCAGCG4ITS4_NexteraGTCTCGTGGCTCGGAGATGTGTATAAGAGACAG5TCCTCCGCTTATTGATATGC
The PCR recipe was as follows:   of diluted template DNA,   of forward primer (10 M),   of reverse primer (10 M),   of nuclease-free water,   of 5X KAPA HiFi Buffer,   of KAPA dNTPs mix (10mM), and   of KAPA HiFi Hot Start Polymerase,   DMSO,   ROX dye (25mM), and   1000x SYBR Green dye. We amplified samples using the following cycling conditions:   for  ; 20 cycles of   for  ,   for  , and   for  .
?. Check PCR products with Agarose gel electrophoresis (optional).
?. Pool PCR duplicate samples together.
?. Second PCR is conducted for attaching standard illumina handles and index primers.  AB1The primer sequences2Forward indexing primerAATGATACGGCGACCACCGAGATCTACACXXXXXXXX3TCGTCGGCAGCGTC4Reverse indexing primerCAAGCAGAAGACGGCATACGAGATXXXXXXXXGTCTC5GTGGGCTCGG
AB1The primer sequences2Forward indexing primerAATGATACGGCGACCACCGAGATCTACACXXXXXXXX3TCGTCGGCAGCGTC4Reverse indexing primerCAAGCAGAAGACGGCATACGAGATXXXXXXXXGTCTC5GTGGGCTCGG
The PCR recipe was as follows:   of diluted template DNA,   of forward primer (10 M),   of reverse primer (10 M),   of nuclease-free water,   of 5X KAPA HiFi Buffer,   of KAPA dNTPs mix (10mM),   DMSO, and   of KAPA HiFi Hot Start Polymerase. We amplified samples using the following cycling conditions:   for  ; 10 cycles of   for  ,   for  , and   for  ; and then a final extension at   for  .
?. All mixtures were purified using Agencourt AMPure XP (Beckman Coulter).
?. Pool the PCR samples in equal DNA amount (ng) or for unequal length amplicons, in equal molecule amount (mol).
?. The pooled samples were size selected at 427 bp ± 20%, denatured with NaOH, diluted to 8 pM, and then spiked with 15% PhiX.
?. Load the denatured, diluted pooled library into the loading position of the Illumina reagent cartridge (V3, 600 cycle kit). Load reagent cartridge, flow cell, and PR2 buffer into Miseq instrument, confirm the metrics and start the run.
TITLE: Rodent Facility Environmental Summary
AUTHORS: Debra Dukes, Michael Elmore

[DESCRIPTION]
Mice (Mus Musculus) are the most common research organisms used in biomedical research. We have 
housed and maintained a variety of strains at the Stowers Institute for Medical Research since 2000.
Our environment and housing parameters provide a standardized guideline for our staff to follow.
This information assures the protection of our research using the rodent model, with proper use of 
housing, nutrition, maintenance, and environmental enrichments for the healthiest rodent specimens
for our research. The provided guideline is currently being used at the Stowers Institute in 2023.

[STEPS]
1. Rodent Facility Environmental Summary
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol has been optimized for electroporation of natural communities in coastal surface waters. As natural communities may vary in different places, we recommend to use this protocol as a starting point and re-optimize according to the results. Guidelines are provided. </p>

[BEFORE_START]
<p>It is really important to carefully plan the sampling of sea water taking tidal and wave conditions into account. The abundance of the sea water communities may be reduced when samples are taken in periods of rough tides. </p>
<p> </p>
<p>To increase the recovery of cells, make sure to use large volumes of sea water (50 liters). Samples can be stored overnight to be processed the day after, but we do not recommend using sea water samples kept for more than 2 days.</p>
<p> </p>
<p> </p>
<p> </p>

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