eamag/protocols-with-steps huggingface dataset:
TITLE: Ikaria Lean Belly Juice Uk Reviews
AUTHORS: lkarialeanbellyreviews  

[DESCRIPTION]
Ikaria Lean Belly Juice Uk Reviews


[STEPS]
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Still, try Ikaria Spare Belly Juice, and be a part of the happy guests, If you're looking for a threat-free weight loss protagonist. Before spending your plutocrat on it, read this detailed review of Ikaria Spare Belly Juice and see if it fits your conditions. 

Ikaria Spare Belly Juice Reviews 

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Official Website:- https://urbanmatter.com/ikaria-lean-belly-juice-reviews-is-ikaria-juice-canada-legitimate/
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>RNA samples (RNA concentration: 100 ng/mL) were extracted from the kidney tissues of both homozygous knockout and wild-type mice and subjected to gene array analyses (Agilent Technologies, Santa Clara, CA) (GEO array GES57590). RNA samples were extracted from the blood of healthy subjects and patients with OPLL, and then subjected to microRNA array analyses (Agilent Technologies, Santa Clara, CA) (GEO array GES57592). Total RNA was isolated from frozen whole tissue samples with TRIzol (Invitrogen, Carlsbad, CA, USA), and from blood samples with Blood RNA Tubes (PAXgene; PreAnalytiX, Hombrechtikon, Switzerland). RNA samples with RIN values ≥ 9.0 as evaluated using a 2100 Bioanalyzer (Agilent, Waldbronn, Germany) were selectively used for these analyses. Pathway analysis was performed based on results of genomic and miRNA arrays (Agilent Technologies, Santa Clara, CA, USA) of samples from patients with OPLL.</p>

[STEPS]
TITLE: Anti-GFP pull-down with syringe filter partition
AUTHORS: Dorothy zhao

[DESCRIPTION]
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease
protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

[STEPS]
SECTION: Sample lysis and filter partition
1. Htt64Q-GFP induced with Muristerone A for polyQ expression in Neuro2a, for two days in a 15cm dish 
SECTION: Sample lysis and filter partition
2. wash with PBS, and lysed in cold isotonic buffer (0.25M sucrose, 1mM EDTA, 20mM HEPES pH 7.4) with protease inhibitor, with a gauge 22-needle for 10 times.
SECTION: Sample lysis and filter partition
3. Nuclei were pelleted at 800g for 10min at 4°C
SECTION: Sample lysis and filter partition
4. supernatant was then centrifuged at 16000g for 10min at 4°C to pellet aggregates and the associated proteins
SECTION: Sample lysis and filter partition
5. The pellet was washed once in PBS to remove soluble Htt64Q-GFP and was then dissolved in PBS, and passed through pre-equilibrated syringe filters (in the same buffer) with 0.2um or 0.4um pore sizes (Millipore). 
SECTION: Sample lysis and filter partition
6. The filter blocked fractions were then collected by washing it with a syringe, in the same buffer, and pelleted again at 16,000g for 10min at 4°C.
SECTION: Anti-GFP pull down
7. The fractions were then incubated with magnetic GFP-Trap (ChromTek) for 6 h at 4°C in binding buffer (140 mM NaCl, 25 mM Tris pH 7.6–8.0, 0.5% NP40, 1 mM EDTA) and 2% BSA (Sigma). 
SECTION: Anti-GFP pull down
8. Lysates from cells without muristerone A induction was used as negative control in pull down. 
SECTION: Anti-GFP pull down
9. After 5 washes with the binding buffer, the bound fraction was eluted and denaturation in sample buffer for dot blot analysis.
TITLE: Morphometry of thyroid cartilage, epiglottis and pyriform fossa
AUTHORS: Murlimanju Virupakshamurthy

[DESCRIPTION]
Larynx is a complex organ of voice production and respiration, which is supported by a series of cartilages, membranes, muscles and joints. They bring about movement of vocal cords with a considerable range of mobility. Apart from the phonating mechanism, it also serves as a sphincter or watch dog of the lower respiratory tract. The knowledge of laryngeal anatomy is important to various professionals such as phoniatricians, speech therapists, oncologists and otorhinolaryngologists. Understanding the laryngeal framework is mandatory in constructing biomechanical models for voice disorders that requires accurate
laryngeal dimensions. The data of the dimensions of thyroid cartilage, epiglottis and piriform fossa are
essential to the anesthesiologists during the laryngoscopy and endotracheal intubation. However, they are scarce in the scientific literature. There are not many studies available about the measurements of thyroid cartilage, piriform fossa and epiglottis in our sample population.

[GUIDELINES]
This study was approved by the ethics committee of our institution (Approval
Committee Name: Institutional Ethics Committee, Kasturba Medical College,
Mangalore, Approval Number: IEC KMC MLR: 02/2022/61, dated 17.02.2022)

[STEPS]
SECTION: Morphometry of thyroid cartilage, epiglottis and pyriform fossa
1.     The goal of this study will be to obtain
the dimensions of thyroid cartilage, epiglottis and piriform sinus in embalmed
cadavers of Indian sample population.
TITLE: Single nuclei RNA sequencing (snRNA-seq) of frozen human lung tissue and hPCLS
AUTHORS: Heidi Monroe, Nayra Cardenes, Oliver Eickelberg, Melanie Königshoff, koenigshoffm, Robert Lafyatis

[DESCRIPTION]
Single-cell RNA sequencing (scRNA-seq) has become an essential tool for delineating cellular diversity in normal tissues and alterations in disease states. This technique requires the dissociation of tissue specimens into cell suspensions. However, the isolation of intact cells can be challenging due to factors such as fragility, large size and tight interconnections. Additionally, single-nuclei isolation can be performed on frozen tissue, enabling the analysis of biobanked samples in a single batch. This protocol for single-nuclei RNA sequencing (snRNA-seq) provides an alternative approach to scRNA-seq, overcoming these limitations to generate high-quality transcriptomic data.

The analysis of gene expression at the cellular level has proven to be a powerful tool for understanding various aspects of lung biology and disease, particularly the process of lung aging. Aging affects different cell types within the human lung heterogeneously, leading to a range of associated changes in their function. Examining the patterns of gene expression in the numerous lung cell types provides insights into the aging and disease processes that contribute to cellular dysfunction. By analyzing these changes at the single-cell level, we can delineate the complex cellular diversity in the human lung and track alterations in molecular pathways involved in the dynamic process of lung aging. Understanding these gene expression patterns will offer opportunities for timely interventions and the identification of biomarker for early prognosis and personalized treatment therapies.

This protocol collection describes the process of single nuclei RNA sequencing from nuclei isolation from frozen tissue (whole, or from PCLS), to barcoding, library construction and sequencing.

[STEPS]
TITLE: Rapid Run v2 Primer Rehyb
AUTHORS: Amanda  Chilaka, Sumeet  Gupta

[DESCRIPTION]
Switch the flowcell to another sequencer if first base fails - HiSeq

[GUIDELINES]
If a rapid run has failed at first base and you want to switch the flowcell to another side or sequencer, follow these steps (ONLY applicable to single read runs, changing sequencer for paired end run is not possible)

These steps are to be performed on the HiSeq where the flow cell is being moved.

[STEPS]
SECTION: Rapid Run v2 Primer Rehyb
1. 
Wash the sequencer that will be used (MWB and then water). [Link to wash protocol]
SECTION: Rapid Run v2 Primer Rehyb
2. Load the SBS rack (with reagents) to the sequencer.
SECTION: Rapid Run v2 Primer Rehyb
3. Load water in the Paired End rack and the template tubes.
SECTION: Rapid Run v2 Primer Rehyb
4. Place a dummy flowcell on the deck. Engage vacuum.
SECTION: Rapid Run v2 Primer Rehyb
5. Setup the run as normally you would, but select “cluster generation on cbot.” even if it was onboard clustering.  This will save an additional hour of time.
SECTION: Rapid Run v2 Primer Rehyb
6. Enter the other parameters used for the flowcell, such as read lengths, flowcell ID, etc...This will create the files needed to allow you to do a rehyb later.
SECTION: Rapid Run v2 Primer Rehyb
7. Start the run and wait for the first base report to come up. End the run.
SECTION: Rapid Run v2 Primer Rehyb
8. Thaw a Rehybridization kit and replace or top-off the reagents according to the “HiSeq Rapid Primer Rehybridization Reference Guide.” A general summary is below.
SECTION: Rapid Run v2 Primer Rehyb
8.1. For Single-Read Runs, replace/top-off the following reagents with these from the Rehyb kit:
 
     Position      Reagent      Description        5      USB      Add   to current USB bottle        18      HP10      Replaces   Read1 primer        17      HP12      Replaces   Index1 primer (i7)        16      HP9      Replaces   Index2 primer (i5)   
 
SECTION: Rapid Run v2 Primer Rehyb
9. Leave the dummy flowcell on the sequencer. You will prime following reagent twice: 
FDR (position 15) - aspiration rate of 1500, dispense rate of 2000, and using 250uL
Flow USB (5) - aspiration rate of 1500, dispense rate of 2000, and using 250uL 
14. 
SECTION: Rapid Run v2 Primer Rehyb
10. Return to the Main screen and select “Sequence,” then “Rehyb Run.” 

Replace the dummy flowcell with sequencing flowcell and proceed with rehybridization.
TITLE: Intracellular metabolomics extraction
AUTHORS: Juan  Sanchez

[DESCRIPTION]
This quench and intracellular extraction are optimal for the analysis of metabolites stable at high temperatures and moderately acidic pH that are rapidly consumed such as pyruvate and acetyl-CoA. It also avoids the use of methanol which can form degradation products with many metabolites of interest. It provides a near-complete extraction of polar and semi-polar metabolites with negligible benefits from re-extraction. It also enables the simultaneous generation of intracellular and extracellular metabolomics samples amenable for stable isotope resolved metabolomics timecourse experiments.  The compound classes extracted are best seen when paired with HILIC chromatography. 

[STEPS]
SECTION: Fast Filtration
1. Harvest cells.
The recommended biomass input for mammalian cells is 10 million cells. For bacteria and yeast, 6 relative ODs are recommended.
SECTION: Fast Filtration
2. Dispense cells onto a 47 mm nylon filter with a 0.2 µm pore size under vacuum in a vacuum filtration assembly. If desired the supernatant may also be collected simultaneously by attaching a 50 ml polypropylene tube to the sand core filter head outlet. Avoid rinsing with fresh media, or water as this can lead to a leaky cell membrane and loss of metabolites.
SECTION: Fast Filtration
3. Turn off the vacuum and remove the nylon filter using tweezers.
SECTION: Fast Filtration
4. Coil the filter and insert it into a 5 ml conical tube. 
SECTION: Boiling ethanol extraction
8. Place the tube in heating blocks pre-heated to 100oC for 12 min. The actual temperature of the liquid inside the tube will not exceed 78oC, which is the boiling point of ethanol.
SECTION: Fast Filtration
5. Seal the tube and submerge it in liquid nitrogen to quench metabolism. Keep in liquid nitrogen until ready to extract.
SECTION: Boiling ethanol extraction
7. Dispense 3ml of a 75:25, v/v solution of ethanol and water with 0.1M formic acid per sample tube.
SECTION: Fast Filtration
6. To collect the filtered extracellular fraction disassemble the filter flask assembly, remove the 50 ml conical tube, cap the tube, and store it at 4oCelsius for immediate analysis or extraction.
SECTION: Boiling ethanol extraction
9. Sonicate the tubes with a probe until a homogeneous mixture is achieved.
SECTION: Boiling ethanol extraction
10. Place the tubes in an ice bath for 60 min for protein precipitation.
SECTION: Boiling ethanol extraction
11. Centrifuge the tubes for 10 min at 4K rpm and 2oC.
SECTION: Boiling ethanol extraction
12. Transfer the supernatant in 1ml fractions to a PALL 0.2um GHP deep well filter plate with a 96 deep well-receiving plate attached.
SECTION: Boiling ethanol extraction
13. Centrifuge the plate for 10 min at 4K rpm and 2oC.
SECTION: Boiling ethanol extraction
14. Place the plate in a centrifugal evaporator until dry at 30oC.

SECTION: Boiling ethanol extraction
15. Immediately prior to analysis re-suspend and reconstitute all the dried sample fractions into a single sample using a single 100 ul volume of solution compatible with the mobile phase used.
TITLE: Mmolecular COVID-19 Extraction-Free Direct-One-Step Fast Cycling Protocol
AUTHORS: steve.puts 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Direct One-Step RT-qPCR COVID-19 Test Kit is designed for quantitative</div><div class = "text-block">real-time analysis of target RNA directly from, swabs without the requirement of any prior</div><div class = "text-block">RNA purification steps.</div><div class = "text-block">Shipping: shipped on blue ice </div><div class = "text-block">Storage Conditions: store at -20 °C </div><div class = "text-block">Additional Storage Conditions: avoid freeze/thaw cycles stable at 4 °C for up to 4 weeks </div><div class = "text-block">Shelf Life: 12 months</div><div class = "text-block">Form: liquid / lyophilized</div></div>

[STEPS]
?. [Sample processing]
Place the swab brush into a 1.5ml microcentrifuge tube containing  and .Rotate the brush 5-10 times.Squeeze the brush and remove it from the tube.
[PCR-grade water]
[PBS (10x conc)]
?. [Sample processing]
Centrifuge at   for 3 min at room temperature
Centrifuge: 12000 34
?. [Sample processing]
Discard the supernatant.Add  and Briefly flick the sample.
[PCR-grade water]
[Extraction Buffer]
?. [Sample processing]
Incubate for 3 min at room temperature for lysis and release of RNA.
?. [Sample processing]
Centrifuge briefly and transfer  to the well plate.Pipette up/down to make sure the sample has fully released from the pipette.
[sample]
?. [Sample processing]
Cover the sample with
[mineral oil]
?. [Cycling]
Load the well plate into the cycler and move the chamber door down.Click run.Run with parameters:- RT 50C for 3 min- Denaturation 95C for 0s (maximum speed)- Annealing 60C for 0s (maximum speed)
?. [Well plate preparation]
Pre-mix the reagents for 81 wells
[total volume]
[Direct reaction mix]
[Enzyme mix]
[Primers and probes mix]
[PCR grade water]
?. [Well plate preparation]
Pipette the mix onto the 81 well plate.To gain speed, use a pipette-ing robot.
TITLE: Spotlight highlights - discussing peer-review
AUTHORS: Gabriel  Gasque

[DESCRIPTION]
This protocol offers snipets of interviews with PLOS ONE Lab Protocol authors discussing the motivations to peer-review their protocols, the significance of the peer-reviewer feedback they got, and the implications that these articles have had for their careers.

For the full interviews, visit the protocols.io Spotlight Youtube Channel, and to read the protocols visit he Spotlight Workspace.

[STEPS]
SECTION: Motivation for peer-reviewing protocols
1. Danelle Agnew - Macquarie University, AUSTRALIA
 

SECTION: Motivation for peer-reviewing protocols
2. Jacopo Cerasoni- Loyola University, USA
 

SECTION: Motivation for peer-reviewing protocols
3. Noah Langenfeld - Utah State University, USA
 

SECTION: Motivation for peer-reviewing protocols
4. Simon Tröder - University Hospital Cologne, GERMANY
 

SECTION: Motivation for peer-reviewing protocols
5. Sonsiray Alvarez-Narvaez - United States Department of Agriculture, USA
 

SECTION: Peer-reviewer feedback
6. Danelle Agnew - Macquarie University, AUSTRALIA
 

SECTION: Peer-reviewer feedback
7. Monica Trujillo and John Dennehy - City University of New York, USA
 

SECTION: Peer-reviewer feedback
8. Noah Langenfeld - Utah State University, USA
 

SECTION: Peer-reviewer feedback
9. Simon Tröder - University Hospital Cologne, GERMANY
 

SECTION: Peer-reviewer feedback
10. Sonsiray Alvarez-Narvaez - United States Department of Agriculture, USA
 

SECTION: Significance for researcher&#x27;s career
11. Danelle Agnew - Macquarie University, AUSTRALIA
 

SECTION: Significance for researcher&#x27;s career
12. Fábio da Costa - Universidade Federal do Rio de Janeiro, BRAZIL
 

SECTION: Significance for researcher&#x27;s career
13. Jacopo Cerasoni- Loyola University, USA
 

SECTION: Significance for researcher&#x27;s career
14. Monica Trujillo and John Dennehy - City University of New York, USA
 

SECTION: Significance for researcher&#x27;s career
15. Monica Trujillo and John Dennehy - City University of New York, USA

 

SECTION: Significance for researcher&#x27;s career
16. Noah Langenfeld - Utah State University, USA
 

SECTION: Significance for researcher&#x27;s career
17. Simon Tröder - University Hospital Cologne, GERMANY
 

SECTION: Significance for researcher&#x27;s career
18. Sonsiray Alvarez-Narvaez - United States Department of Agriculture, USA
 

SECTION: Resources
19. Spotlight Youtube Channel

Spotlight Workspace
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Freezing Adherent Cell Lines
AUTHORS: Carolina Lopez

[DESCRIPTION]
This protocol describes how to freeze Adherent cells. Examples of these cells are: A549 cells, LLCMK2 cells, and MDCK cells.

[STEPS]
SECTION: Freezing Adherent cells
1. 
Wash flask 2x with sterile PBS.
Add 2mL of trypsin/T75. Incubate at 37 oC for 2-3 mins or until cells are detached from the flask.
Add 8mL of cell culture media and pipette up and down. Transfer all the media to a 15mL tube. 
Centrifuge at 1200 rpm for 5 min. 
Discard supernatant and resuspend cell pellet in 1mL of cell culture media. Add 9mL of media and pipette up and down to homogenize. 
Count the cells. You will need your cell concentration/# for step 6.
Spin the tube containing the cells in medium again at 1200 rpm for 5 min. 
Meanwhile, prepare the freezing medium. The amount of freezing medium to prepare should be the amount needed to dilute the cell pellet so that you have a final concentration of 1*106 cells/mL. 
Discard the supernatant and resuspend the pellet in 1mL of freezing medium. Add the remaining volume of the freezing medium. Make sure you homogenize the solution well. 
Add 1mL of freezing medium containing 1*106 cells/mL to cryotubes (caps screw from the outside). Label all the tubes with the following information:
Cell Name
Generation/Passage
Date
Name
Cell concentration (if there is space)
11. Transfer the cells to the Stratagene chamber at 4C and then transfer the chamber to the -80C for 1 day. After 1 day, transfer the tubes to the liquid nitrogen. You can keep some vials in the -80C. These could last for at least 2-3 years.
TITLE: PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
SECTION: For generic primers
1. PCR mixture PCR condition for generic primers
 

SECTION: For generic primers
2. PCR condition for generic primers

 

SECTION: For barcoded primers
3. PCR mixture for barcoded primers

 

SECTION: For barcoded primers
4. PCR condition for barcoded primers
TITLE: IHC_cFOS+Parvalbumin
AUTHORS: Bilge Buyukdemirtas

[DESCRIPTION]
Immunohistochemistry protocol for c-FOS and Parvalbumin co-staining of mouse brain tissue slices (30 microns thick).

[GUIDELINES]
All shaker incubations are done at 200 rpm speed.

[STEPS]
SECTION: Staining
1. Antigen Retrieval
Remove PBS from wells. Add 500ul 0.3% citrate buffer into each well.
SECTION: Staining
1.1. Incubate at RT on shaker for 30 min
Incubate at 65 °C for 45 min
Cool down on the bench for 20 min


SECTION: Staining
2. 3X PBS Washes
SECTION: Staining
2.1. Remove citrate buffer from wells and replace with 500ul 1X PBS (or use a dropper pipette and fill the well halfway)
SECTION: Staining
2.2. Incubate at RT on shaker for 15 min  

SECTION: Staining
3. Block Non-Specific Signal
Make enough: 10% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS for all samples (500ul per well)
SECTION: Staining
3.1. Remove PBS and replace with blocking reagent 500ul per well.

SECTION: Staining
3.2. Incubate on shaker RT for 60min. 

SECTION: Staining
4. Primary Antibody 
Make enough: mouse anti-parv (1:1000, Sigma-Aldrich, #P3088) and rabbi anti-cfos (1:1000, cell Signaling, #2250S) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
SECTION: Staining
4.1. Replace the blocking reagent with primary antibody mix, 500 ul per well, and incubate at 4 °C 
  

SECTION: Staining
5. DAY 2
3X PBS Washes
SECTION: Staining
5.1. Remove primary Ab mix from wells and replace with 500ul 1X PBS (or use a dropper pipette and fill the well halfway)
SECTION: Staining
5.2. Incubate at RT on shaker for 15 min  

SECTION: Staining
6. Secondary Antibody 
Make enough: goat anti-mouse AF-555 (1:500, Invitrogen, #A-21422) and biotin goat anti-rabbit (1:1000, Vector Labs, #PK-6101) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
SECTION: Staining
6.1. Remove PBS from wells and replace with 500ul Secondary antibody mix. Cover with aluminum foil. Note: secondary antibodies are light sensitive, so from this point on the plate MUST be covered from light as much as possible. 

Incubate at RT on shaker for 120 min. 

SECTION: Staining
7. 3X PBS washes as described in Step 5 and 5.1 with the plate covered    

SECTION: Staining
8. Tertiary Antibody (for cFOS only)
make enough: AF-488 streptavidin conjugated (1:1000, Thermo Fisher, #S11223) in 1% NGS (Vector Labs, #S-1000-20) in 0.3% triton-X PBS
SECTION: Staining
8.1. Replace PBS in wells with 500uL of tertiary antibody solution, cover the plate with aluminum foil, and incubate at RT on shaker for 120 min.
SECTION: Staining
9. 3X PBS washes as described in step 5 and 5.1 with the plated covered   

SECTION: Mounting
10. Mounting with medium with and without DAPI
TITLE: Isolation and Characterization of Immune Cells from the Tumor Microenvironment of Genetically Engineered Pediatric High Grade Glioma Models Using the Sleeping Beauty Transposon System
AUTHORS: Maria B Garcia Fabiani, Andrea Comba, Padma Kadiyala, Santiago Haase, Felipe Javier Núñez, David Altshuler, Pedro R Lowenstein, Maria G Castro

[STEPS]