eamag/protocols-with-steps huggingface dataset:
TITLE: Storage and Processing of Tissue for bulk RNA Isolation
AUTHORS: Aaron Horning

[STEPS]
?. [Storing Tissue for RNA Isolation]
After resection, tissue is immediately flash frozen in 50 ml Falcon tube or 2 ml cryovial tubes by placing tissue into tube then simply dropping filled tube into liquid nitrogen.
?. [Storing Tissue for RNA Isolation]
The tubes are stored on dry ice or -80C until they can be stored in a liquid nitrogen tank
?. [Bulk RNA Isolation]
The large tissue pieces are smashed in a freezing cold mortar and pestle with liquid nitrogen. The desired amount of tissue (30mg - 60mg) is then pulverized and swept into a 1.5 ml centrifuge tube sitting on dry ice to prevent thawing of the tissue.
Its ciritically important for RNA integrity to not let the tissue thaw. Thawing will activate the endogenous RNases within the tisses thus depleting the chemical we wish to assay.
?. [Bulk RNA Isolation]
Follow the Quiagen All Prep Kit protocol found here: https://www.qiagen.com/us/resources/resourcedetail?id=bbd50261-3b80-4657-ad58-6a5a97b88821&lang=en. Complete the RNA isolation steps.
?. [Bulk RNA Isolation]
Breifly, be sure to lyse and homogenize the tissue with RLT buffer with beta-mercaptoethanol with an 18 gauge needle until the needle runs smoothly. Additionally, if once the lysed tissue in RLT appears to be at all yellow and not clear, dilute the lysed tissue in RLT in more RLT buffer. If the RLT is too dense, it will not run through the columns and you'll loose material.
?. [Bulk RNA Isolation]
Elute in water and freeze at -80C.
TITLE: Microorganisms and culture conditions - Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis (Rocha et al., 2019)
AUTHORS: Lis Rocha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between four strains of </span><span style = "font-style:italic;">S. aureus</span><span> causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122, especially in genes encoding host immune evasion and surface proteins. The comparison of orthologous genes using OrthoMCL identified a membrane transporter in the genomes of the bacteria belonging to the subclinical group, but this finding was not confirmed by polymerase chain reaction (PCR) on a collection of field isolates of </span><span style = "font-style:italic;">S. aureus</span><span> associated with clinical or subclinical mastits. The secreted and surface proteins predicted by different in silico tools were compared through multidimensional scaling analysis, revealing a high degree of similarity among the six strains. However, differences were seen in the nucleotide sequences of a gene that codes for a hypothetical protein (cl3309) and a lipoprotein (cl3700). These findings were also analyzed by PCR on DNA extracted from field isolates of</span><span style = "font-style:italic;"> S. aureus</span><span>. The lipoprotein, but not the hypothetical protein, was able to separate the clinical isolates from the subclinical ones. These results show that sequence variation among bovine </span><span style = "font-style:italic;">S. aureus</span><span>, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes</span></div></div>

[STEPS]
?. [Growing and storing conditions]
The bacteria used in this study were maintained on BHI agar (Brain Heart Infusion, BHI HiMedia, Mumbai, India) at 37 °C
?. [Growing and storing conditions]
and stored in the long term in BHI containing 20% glycerol.
TITLE: Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i</span><span style = "vertical-align:super;">3</span><span>Neurons) or lower motor neurons (i</span><span style = "vertical-align:super;">3</span><span>LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i</span><span style = "vertical-align:super;">3</span><span>Neurons or i</span><span style = "vertical-align:super;">3</span><span>LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc. </span></div></div>

[STEPS]
TITLE: Single digested
AUTHORS: Jiaxin Li

[STEPS]
?. Add the following reagents to a tube.  AB1Total10ul210&nbsp;x&nbsp;Buffer1ul3Template（500ng）According to different template4Enzyme0.5ul5ddWaterAdd to 10ul
AB1Total10ul210&nbsp;x&nbsp;Buffer1ul3Template（500ng）According to different template4Enzyme0.5ul5ddWaterAdd to 10ul
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 1.5 hours (according to different enzymes)
TITLE: Staging colonial ascidians
AUTHORS: Simon  Blanchoud, Marta   Wawrzyniak

[DESCRIPTION]
This protocol has been successfully used to stage Botrylloides diegensis in our laboratory and was based on the following publication:
Ontology for the Asexual Development and Anatomy of the Colonial Chordate Botryllus schlosseri 

[STEPS]
SECTION: Staging
1. Select the slides to be staged.
SECTION: Staging
1.1. Clean the colony. See Cleaning colonial ascidians V.2
SECTION: Staging
3. Observe the organization of the zooids and the systems. Check for signs of takeover:
SECTION: Staging
3.1. No signs of active filter-feeding.
SECTION: Staging
3.2. Zooids within systems are not properly fused yet.
SECTION: Staging
3.3. Remnants of resorbed zooids are still visible.
SECTION: Staging
4. Turn the colony upside-down. Look for buds to stage.
SECTION: Staging
4.1. Start at low magnification, look for well-visible buds (the one that are not too much underneath the zooid-parent, typically posterior to the adult).
SECTION: Staging
4.3. Monitor the activity of the heart. Check for signs of regular beating:

Flow will be flowing through the bud at all stages, even without heart beats.
At early stages, heart will be erratic and weak.
Every minute or so, flow will be reversed and heart beat will stop transiently.

SECTION: Staging
5. Look for secondary budlets to stage.
SECTION: Staging
5.1. Budlets are often hidden or difficult to observe, focus on the most evident ones.
SECTION: Staging
5.2. Variability can be observed within a colony. Monitor multiple buds and use the average stage as your reference.
SECTION: Staging
2. Fill the Petri dish with ASW. See Artificial sea water  V.2
SECTION: Staging
7. The best method to ensure the current stage is to confirm it by staging on consecutive days. Stages should proceed approximately by one per day (at 20°C).
SECTION: Staging
6. Use the provided identification scheme to infer the stage from your observations.   

SECTION: Staging
8. Take a couple of representative images for archiving.

SECTION: Staging
4.2. Once the buds are identified, zoom in.
TITLE: Pulling Pipettes using the DMZ-Zeitz Universal
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol is used for the manufacture of glass pipettes for patch clamp electrophysiology using the DMZ-Zeitz Universal puller.</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: Sectioning Rat Heart
AUTHORS: Shaina Robbins, Sean Nieves

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the straightforward procedure we follow when sectioning rat heart.</div></div>

[STEPS]
?. [Sectioning]
1. Take rat heart our of  freezer and place into cryostat.2. Set the cryostat to desired tissue thickness and begin the tissue sectioning process3. Collect the tissue section gently onto the slide4. Immediately after tissue sections are collected, place slides into a box containing dry ice until slides can be transferred to the  freezer.
-80 °C
Allow heart sample to sit in cryostat for at least 20-30 minutes before beginning to section. This will allow for the OCT to equalize to the cryostat temperature and prevent any cracking in the OCT and tissue to occur.
For rat hearts typically two sections are collected per slide. A variety of slide types (superfrost glass +, histobond, frame membrane and glass membrane) can be used.
-80 °C
TITLE: Dot blot to verify microinjection efficacy in Euplotes crassus
AUTHORS: RACHELE CESARONI, Rachele Cesaroni

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

[STEPS]
?. Grow cells up to a density of 1000 cells/ml, and transfer 400 μl of each culture to an Eppendorf tube.
?. Pellet the cells at max speed for 5 minutes, and resuspend them in 400 μl of ddH2O.
?. Add 50 μl of 0.5 M EDTA, pH 8.0 and 50 μl of 4 M NaOH to the cells.
?. Lyse the cells at 68°C for 30 minutes, and centrifuge them for 1 minute at 13000 rpm, then chill them on ice.
?. Soak a membrane for nucleic acid binding and a sheet of Whatmann paper in 0.4 M of NaOH, and place both in a Dot blot apparatus.
?. Switch on the vacuum pump, and transfer all the lysates into the chamber.
?. Soak the membrane in 0.4 M NaOH for 15 minutes, and wash it in 2X SSC buffer for 10 minutes, then drain it on a sheet of Whatmann paper.
?. Pre-hybridize the membrane in 20 ml of Church buffer at 60°C for 2 hours, then hybridize it at constant temperature (annealing temperature) o/n in 10 ml of Church buffer containing the radioactive probe.
?. Wash the membrane twice with 2X SSC, 0.1% SDS for 15 minutes each time.
?. Place the membrane in a plastic foil, expose it overnight and scan it to determine which clones have sufficient DNA from the microinjection (compared to suitable DNA standards used as positive controls).
TITLE: Genomic DNA extraction from Mycobacterium avium
AUTHORS: Tetsuya Komatsu, Kenji Ohya, Justice  Opare Odoi, Shota  Suganuma, Kotaro Sawai, Takayuki Wada, Tomotada Iwamoto, Fumito Maruyama

[DESCRIPTION]
Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in human and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. Here, we isolated  MAH from pig lymph nodes or livers and obtained their genomes.

[STEPS]
1. Suspend colonies on 7H11 agar into 300 µl of TE.
2. Centrifuge at 5,000 g for 10 min.
3. Discard supernatant, followed by suspension in 300 µl of acetone.
4. Centrifuge at 5,000 g for 10 min.
5. Discard acetone, followed by suspension in 200 µl of TE.
6. Add lysozyme at the final concentration of 100 µg/ml.
7. Incubate at 37°C overnight.
8. Add proteinase K at the final concentration of 0.2 mg/ml.
9. Incubate at 50°C for 3 hrs.
10. Add 1/10 volume of 3 M NaOAc.
11. Add equal volume of phenol/chloroform/isoamyl-alcohol and shake vigorously.
12. Centrifuge at 12,000 g for 10 min.
13. Transfer aquatus phase to a new tube.
14. Add equal volume of 2-propanol.
15. Centrifuge at 12,000 g for 10 min.
16. Remove supernatant and wash with 70% ethanol.
17. Suspended in 400 µl of TE, followed by adding RNase at the final concentration of 100 µg/ml.
18. Incubate at 37°C for 30 min.
19. Repeat steps 10 to 16.
20. Suspended in 100 µl of TE.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to concentrate viruses in liquid samples using an Amicon or Nanosep centrifugal ultrafiltration device. We use Amicons to concentrate medium volumes of samples (10s to 100s of mls) down to a final volume of ~4 ml. We use Nanoseps to concentrate smaller volumes of sample (&lt;10ml) down to a final volume of ~40&micro;l.

[STEPS]
?. 
?. 
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?. 
?. 
?. 
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?.
TITLE: Removal of the Female Reproductive System En Bloc
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes removal of the ovaries, Fallopian tubes, and uterus as a whole from the pelvis.

[STEPS]
1. Identify right round ligament and suture ligate laterally with 2-0 vicryl (Figure 1a).

  

2. Divide right round ligament and continue dividing broad ligament cephalad toward the vesicouterine peritoneal reflection using Metzebaum scissors.
3. Similarly identify, ligate, and divide left round ligament and incise broad ligament cephalad toward the cervix to meet the right side (Figure 1b).
4. Using two fingers, push bladder cephalad to separate it from the uterus and cervix. 

Note: Prior cesarean deliveries can make this difficult.
5. Identify, clamp, and cut right and left ovarian arteries and veins (also called the infundibulopelvic ligaments) using curved clamps and Mayo scissors (Figure 1c, 1d).
6. Divide the broad ligament laterally, keeping ovaries and Fallopian tubes medial to and attached to the uterus using scalpel or Mayo scissors (Figure 1e).
7. Further mobilize bladder off the cervix and proximal vagina with blunt dissection.
8. Clamp and cut the uterine artery and vein and surrounding parametrium bilaterally.
9. Clamp and cut the uterosacral ligaments (Figure 1f, 1g).
10. Palpate the bottom of the cervix between the bladder and rectum using a thumb and forefinger.
11. Place two curved Heaney clamps beneath the cervix across the vagina.
12. Use a scalpel or Statinsky scissors to transect vagina and remove FRS en bloc from the pelvis.
13. Use Allis clamps to grasp the anterior and posterior walls of the vagina.
14. Close vagina using a running 0-vicryl suture.
15. Obtain pictures of front and back of preservation solution to obtain the lot number and expiration date.
16. Measure organs and place in a bag on ice and prepare for transport, keeping individual organs on ice throughout the following protocols, when not actively working with them (Figure 2).
TITLE: SUPERCAPACITOR
AUTHORS: a

[DESCRIPTION]
en este experimento utilizamos los componentes para crear un circuito rc, posteriormente lo conectamos a un osciloscopio para obtener los resultados

[BEFORE_START]
estar preparados para realizar el experimento con las medidas de seguridad adecuadas

[GUIDELINES]
seguir paso a paso el protocolo que se mencionará a continuacion

[STEPS]
1. Primero analizamos el ejemplo puesto por el profesor de como es un circuito equivalente a un supercapacitor.
2. Tomamos los componentes y aparatos necesarios:
Protoboard
1 capacitor de 10 microFaradios
1 capacitor de 100 microFaradios
2 resistencias de 100 Ohms
1 resistencia de 1000 Ohms
5 cables de cobre
Oscilosopio
Fuente
  

3. Tomamos la protoboard y conectamos en serie las dos resistencias de 100 Ohms, estas seran R1 Y R2
   

4. Conectamos la resistencia de 1000 Ohms en serie con R1
  

5. Alineado al punto donde se unen R1 Y R2 conectamos el positivo de nuestro capacitor de 10 microFaradios (C1) de un lado de la protoboard y el negativo alineado pero en el otro lado
6. Alineado al punto donde acaba R2 conectamos el positivo de nuestro capacitor de 100 microFaradios (C2) de un lado de la protoboard y el negativo alineado pero en el otro lado
  
  

7. Tomamos dos cables y con cada uno de los capacitores, conectamos a la patita positiva un extremo del cable y el otro extremo a la fila negativa de la protoboard.
8. Conectamos otro cable desde la fila positiva de la protoboard, a nuestra resistencia RC.
9. Conectamos en la fila positiva un cable para que sea la terminal positiva de nuestro circuito y otro en la fila negativa para que sea la terminal negativa.
el modelo quedaría algo así:
  

10. Comenzamos con el experimento, conectamos el osciloscopio  a nuestro Capacitor 2, con el negativo al caiman y el positivo al ganchito, nos podemos apoyar de conectar dos cables a los polos del capacitor.
11. Conectamos nuestras terminales positiva y negativa a la fuente.
 

12. Ajustamos la fuente para que de 5 Voltios.
  

13. Ajustamos el osciloscopio para que nos de los datos de una manera adecuada y visible.
14. Descargamos el capacitor tomando ambos extremos de un cable y conectandolo a los polos del capacitor
15. Encendemos la fuente y vamos a ver como en el osciloscopio se muestran unas curvas, al momento en que las curvas se regularicen, detenemos el osciloscopio y tomamos los datos con nuestra usb
  

16. Filtramos los datos en un excel.
17. Tabulamos en matlab nuestros datos.|
TITLE: Overall protocol for top-down LC-MS/MS of human heart tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down LC-MS/MS data on human heart tissue

[STEPS]
SECTION: Tissue Collection
1. Heart tissue was provided by University of Washington. Tissue was collected and prepared according to the following: 
 

SECTION: Sample Preparation
2. Perform LC based Proteomics on adjacent tissue sections:
 

SECTION: Data Acquisition
3. 
 

SECTION: Sample Preparation
4.
TITLE: Statistical Analysis Plan for Validation of the STarTBack Tool for Management of Low Back Pain in the Military Health System
AUTHORS: Daniel I  Rhon, Steve    George, Emily  Poehlein, Cindy    Green

[DESCRIPTION]
DISCLAIMER: The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Army, the Department of Defense, the Uniformed Services University of the Health Sciences, the Defense Health Agency or the U.S. Government.

[STEPS]
TITLE: Glass Milk preparation
AUTHORS: Martin Codyre

[STEPS]
?. [Glass Milk Preparation]
After acid washing for   at room temperature  , silica was pelleted by spinning two minutes at  5,000 xg and the supernatant was poured off.
[Possibly 4 to 8 hours]
0 Room temperature
Centrifuge: 5000 33, 2 min
?. [Glass Milk Preparation]
To prepare glass milk, 325 mesh silicon dioxide (Spectrum Chemicals - SI108) was combined with an excess volume of 10% HCl (~3 N HCl) made from combining 37%and MilliQ water (Millipore) in a fume hood
(dry silica powder should not be inhaled).
?. [Glass Milk Preparation]
The pellet was resuspended in four pellet volumes of  and then pelleted again.This wash step was repeated for a total of six washes.
?. [Glass Milk Preparation]
The pellet was then washed with four pellet volumes of 10 mM Tris HCl, pH = 8 (ThermoFisher Scientific AM9855G) and 1 mM EDTA (ThermoFisher Scientific 15575020), and pelleted.
?. [Glass Milk Preparation]
Finally, the pellet was resuspended in 1 pellet volume of 10 mM Tris HCl and 1 mM EDTA and autoclaved. This autoclave step is likely superfluous, however, as acid washes should render the beads free of contaminants. The resulting 50% glass milk slurry can be stored at room temperature. Before use, care must be taken to vigorously resuspend the particles as they begin to settle quickly.
TITLE: Protcol 1: PCR Dry Lab
AUTHORS:  

[STEPS]
?. [Finding a Sequence of Interest: Human Genome Browser]
To find information on DNA region of interest, first go to the hgb website and find the Genomes tab. Click on SARS-coV-2 (COVID-19).
?. [Finding a Sequence of Interest: Human Genome Browser]
Once the genome is open, go to the bottom of the page and click default tracks. In addition to the default tracks, open the ARTIC primers track under the Mapping and sequencing section and NCBI genes under Genes and Gene Predictions.
?. [Finding a Sequence of Interest: Human Genome Browser]
Using the tracks on display, give the name of a protein that is known to bind with ACE2 and give a description of the protein’s function.
?. [Finding a Sequence of Interest: Human Genome Browser]
Using the tracks on display, give the name of the ORF (open reading frame) in which the protein is translated from. Give the name of the track used to find this information.
?. [Finding a Sequence of Interest: Human Genome Browser]
Once you have your protein of interest, find the specific region on the protein which is known to bind ACE2. This is our region of interest. Give the name of the track that displays what the region of interest does and give the start and stop position of this ACE2 binding region.
?. [Finding a Sequence of Interest: Human Genome Browser]
Using the ARCTIC primer track, find a set of forward and reverse primers that would encapsulate the full region of interest, ACE2 binding region. Give the names of the primers.
?. [Designing Primers and Experimental Amplification: Geneious]
Take a look at an annotated plasmid of SARS-CoV-2, a file can be found on Resources tab of protocols.io. File name is SARS-CoV-2_(covid19)_annotated_plasmid.gb. Open the file on Geneious
?. [Designing Primers and Experimental Amplification: Geneious]
On the plasmid, find the gene, respective to the protein, in which your region of interest is on. Highlight the region on which the gene is located. Click the button labeled “Add Annotation”. Name the annotation and label the correct annotation type. What type of annotation should this protein be labeled as?
?. [Designing Primers and Experimental Amplification: Geneious]
The annotation should display as a block on the plasmid. Click on the annotation block and then click the button labeled “Translate”. Give the single letter amino acid code found in the range of 180 to 190.
?. [Designing Primers and Experimental Amplification: Geneious]
Find the region of interest, ACE2 binding, on the gene you have just annotated. Now annotate the region of interest. Translate the region and give the single letter amino acid code found in the range of 30 to 40.
?. [Designing Primers and Experimental Amplification: Geneious]
Create two sets of primers, one to amplify the protein region and to amplify the region of interest. Give the interval at which the primers exist, base pair length, GC content, and Tm. Make sure primers are up to general standards. Hint: one can check if primer meets criteria by hovering the mouse over the primer.