eamag/protocols-with-steps huggingface dataset:
TITLE: Whole Organoids Harvesting Procedure (Cultrex-modified)
AUTHORS: Gabriela Vallejo Flores, Annika Fendler

[DESCRIPTION]
This protocol is use for whole organoids isolation, after isolation organoiods may be:
a. Resuspended in basement membrane matrix for further organoid culture.
b. Resuspended in freezing medium for cryopreservation.
c. Processed for biochemical analysis (such as RT-PCR, MS-PCR, sequencing, Western Blot, ELISA, or IHC)

[BEFORE_START]
Pre-warm seeding plate
work with 1% BSA/PBS coated tubes and pipettes
use pre-cooled pipette tips for matrigel
precool centrifuge
precool PBS and Cultrex™ Organoid Harvesting Solution
Working on ice

[STEPS]
SECTION: Sample description
1. 
 

SECTION: Organoids harvesting
2. Aspirate cell culture media and gently wash each well with 10 volumes cold (2-8 °C) PBS (Table 2). Be careful not to disrupt the basement membrane matrix containing organoids. 

 

SECTION: Organoids harvesting
3. Add 10 volumes of cold (2-8 °C) Cultrex™ Organoid Harvesting Solution to each well (Table 2). Dislodge the dome with the pipett and transfer to a 15 ml Falcon.
SECTION: Organoids harvesting
4. 
Incubate for 60 min to 90 min, shaking at   on IKA rocker 3D on a cold pack in a Polystyrene box. This incubation is complete when the basement membrane matrix is no longer visible.

SECTION: Organoids harvesting
5. Centrifuge the tube at 500 x g, 5 min, 2-8 °Cin a swinging bucket rotor to pellet the organoids. Aspirate the supernatant.
SECTION: Organoids harvesting
6. Wash organoids with 10 volumes of cold (2-8 °C) PBS (Table 2), centrifuge at 500 x g, 5 min, 2-8 °C  in a  swinging bucket rotor to pellet the organoids. Aspirate the PBS.
SECTION: Cell seeding Mullenders
7. Pre-warm a 48, 24 or 6 wells plate and add to the cell pellet the amount of matrigel described in Table 3. Homogenize the matrigel with the cells and dropped in the center of the well. 

 


SECTION: Cell seeding Mullenders
8.  Flip the plate and incubate 15 min at 37 °C  5%CO2.
SECTION: Cell seeding Mullenders
9. Add 250 µL, 500 µL or 2500 µL  of medium per well.
SECTION: Cell seeding Mullenders
10. 
SECTION: Cell seeding Mullenders
11. 
SECTION: Cell seeding Mullenders
12.
TITLE: DNA Extraction from Water Samples
AUTHORS: Andrew   Dominguez, Yanyan Zhang, Nicole  Pietrasiak

[DESCRIPTION]
This protocol was followed to extract DNA from potable and tailored water samples used to water various turf grasses in a greenhouse study. We closely followed the extraction protocol provided in the Qiagen DNeasy PowerWater Kit with a few modifications to account for low product yield.

[STEPS]
SECTION: Sample Prep
1. If water samples are stored at -80 °C allow time for samples to defrost depending on the volume. Large volumes can take a few hours to defrost.

SECTION: DNA Extraction
2. DNA extraction was completed using   with the following modifications:
In step 1, the specific filters used were   
In step 21, 30 µL of Solution EB was added to increase DNA concentration
Following step 21 the wait time was 5 min before moving on to step 22
Following step 23, DNA extracts were stored at -80 °C 




SECTION: DNA Extraction
2.1. A demonstration for folding a filter and inserting it into the Bead Tube can be found here.
TITLE: HTTM : Illumina library preparation
AUTHORS: Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue

[DESCRIPTION]
Part of the HTTM protocol dedicated to the preparation of Illumina sequencing libraries.

[BEFORE_START]
All steps and master mixes need to be kept on ice  as much as possible. Thermocyclers need to be cooled at 4C before inserting sample plate.  

[STEPS]
SECTION: Libraries
1. Transfer 2.5 µL  of DNA from the DNA extraction plate to a new PCR plate. 

SECTION: Libraries
2. Prepare a fragmentation master mix for 96 samples with : 

 

SECTION: Libraries
3. Add 1 µL of the fragmentation master mix to each well. 

SECTION: Libraries
4. Incubate in a thermocycler with the following protocol : 
15 minat 37 °C
30 minat 65 °C




SECTION: Libraries
5. Add 1 µLof 4µM Nextera (NxT) adaptors to each well.

SECTION: Libraries
6. Prepare a ligation master mix for 96 samples with : 
 

SECTION: Libraries
7. Add 3.5 µLof ligation master mix to each well.

SECTION: Libraries
8. Incubate in a thermocycler with the following protocol : 
30 minat 20 °C
10 minat 65 °C

SECTION: Libraries
9. Prepare a PCR master mix with : 

 

SECTION: Libraries
10. Add 92 µLof PCR master mix to each well.

SECTION: Libraries
11. Split the PCR reaction into 2 different plates (50 µl per plate). 
SECTION: Libraries
12. Incubate each plate in a thermocycler with the following cycles : 
30 sat 98 °C
15 sat 98 °C 
30 sat 72 °C
Repeat from step 2 for 20~25 cycles*
2 min  72 °C






SECTION: Libraries
13. Pool the 2 PCR replicates together in a.
SECTION: Libraries
14. Transfer2 µLof DNA from the pool plate to a new PCR plate.

SECTION: Libraries
15. Add 2 µLof each barcoding primer to the DNA : 
- Nxt_i5_barcoding
- Nxt_i7_barcoding

SECTION: Libraries
16. Prepare a PCR master mix with : 

 
 
 

 
 
SECTION: Libraries
17. Add 44 µL of the PCR master mix to each well of the plate. 

SECTION: Libraries
18. Incubate in a thermocycler with the following protocol : 
30 s at 98 °C
15 s at 98 °C
1 min at 72 °C (no anneal step)
Repeat from step 2 for 7 cycles
2 min at 72 °C






SECTION: Libraries
19. Pool together 5 µL of each sample.

SECTION: Libraries
20. Purify with SPRI beads using a 0.8 ratio. Resuspend with50 µL of molecular grade water.

SECTION: Libraries
21. Proceed with QC and sequencing.
TITLE: Paraffin wax as self-sealing insulation material for seasonal sensible heat storages -  Enhancement of thermal performance - Temperature Records
AUTHORS: Christoph Bott, Ingo Dressel, Peter Bayer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This supplement to the article </span><span style = "font-style:italic;">"Paraffin wax as self-sealing insulation material for seasonal sensible heat storage systems – A laboratory study"</span><span style = "font-style:italic;font-weight:bold;"> </span><span>contains the temperature data recorded in the laboratory for section 2.1 </span><span style = "font-style:italic;">"Enhancement of thermal performance"</span><span>.</span></div></div>

[STEPS]
TITLE: Protocol for Exonuclease VIII, truncated (NEB #M0545)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Exonuclease VIII, truncated efficiently degrades linear dsDNA from 5’ to 3’ direction, leaving supercoiled dsDNA intact.</div><div class = "text-block"><span style = "font-weight:bold;">* Note: </span><span>For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.</span></div></div>

[STEPS]
?. Set-up the reaction as follows:  AB1COMPONENTS 50 μl REACTION2DNAup to 1 µg3NEBuffer 4 (10X)5 µl (1X)4Exonuclease VIII (truncated)1 µl (10 units)5Nuclease-free H2O (NEB #B1500)up to 50 µl
AB1COMPONENTS 50 μl REACTION2DNAup to 1 µg3NEBuffer 4 (10X)5 µl (1X)4Exonuclease VIII (truncated)1 µl (10 units)5Nuclease-free H2O (NEB #B1500)up to 50 µl
?. Incubate at  for .
37 °C
?. Stop reaction by adding EDTA to at least .
?. Heat Inactivation  for .
70 °C
?. To clean up treated samples, we recommend using one of the following steps:a. Column clean up (we recommend the Monarch®PCR & DNA Cleanup Kit, NEB #T1030) orb. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), orc. Performing a phenol/chloroform extraction followed by ethanol precipitation.
TITLE: Safety Assessment and Reporting (Part 7 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 7 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: Protocols for the  assembly and annotation of snake genomes
AUTHORS: Boyang Liu, Liangyu Cui, Zhangwen Deng, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
Background: Snakes are one of the most important wildlife resources and are widely distributed. Bungarus multicinctus is a highly venomous snake and is distributed in central and southern China. In recent years, venomous snake bites have gradually increased. Genomic resources are significant for understanding the evolution of a specie and the molecular mechanisms of toxin production.


Find: Here we present a highly- continuous genome assembly of B. multicinctus. The genome size of B. multicinctus is 1.51Gb. The maximal length of scaffold is 39.68M and the N50 length is 6.55M. The B. multicinctus has a GC content of 37.8%.




[STEPS]
TITLE: Cell growth assay
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

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

[STEPS]
?. Cells were seeded into 96-well plates and cultivated for indicated time periods
?. 20 μl of CellTiter 96® AQueous One Solution Reagent was added to each well.
?. The plates were incubated at 37 °C for 1 h prior to measuring the absorbance of each sample using an iMark™ Microplate Reader (BIO-RAD, Hercules, CA, USA) at a wavelength of 490 nm.
?. Cell viability was then calculated by comparing the absorbance of the treated cells with that of the control cells, with the latter defined as 100%.All samples were assayed in triplicate or quadruplicate for each experiment.
TITLE: Irritability-like behavior testing with the bottle brush
AUTHORS: Adam Kimbrough, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h1>Irritability Protocol</h1></div><div class = "text-block">We use the bottle-brush test to test for irritability-like behavior see previous studies for examples (Kononoff et al., 2018, Kimbrough et al., 2017). The bottle-brush test uses a bottle brush to measure aggressive and defensive responses from the rats. 



</div></div>

[STEPS]
?. Procedure:
Testing usually begins during the dark cycle of the animals under dark conditions with a red light for researchers but may vary depending on the experiment.


A session consists of 10 trials in a clean plastic cage (26.67 cm × 48.26 cm × 20.32 cm; Ancare, Bellmore, NY, USA) with fresh bedding. 


A trial begins with the rat positioned at the back of the cage and the rat should be moved to the back of the cage if not there, trials occurring with the rat starting elsewhere should be disregarded.


The person performing the brushing inserts the bottle-brush into the cage from the front, rotating towards the rats’ whiskers for approximately 3 seconds. The brush should make contact with the rat for approximately a second on the whiskers.


The rotating brush is then retreated to the front of the cage over the course of a second.


The brush then is hung vertically for approximately 2 seconds (without rotating) before being removed entirely from the cage. This step and the previous step allow for the rat to follow the brush to the resting point and bite, paw at and inspect the brush if they choose to.


Removal of the brush from the cage indicates the end of a trial, which should be repeated 10 times. After a 5 second gap the trial is repeated.


During the trial the 2-3 observers identify behavioral responses that are categorized into aggressive and defensive. Each behavior that occurs in a given trial is recorded between the trials on scoresheets. A behavior occurring in an individual trial results in a tally mark for that behavior in that given trial on the scoresheet.


At the end of the sessions the total score for each behavior (and aggressive or defensive behaviors) are tallied for each rat and the average across observers is taken.
TITLE: Isolation of Mononuclear Cells from Whole Blood by Density Gradient Centrifugation
AUTHORS: STEMCELL Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to isolate mononuclear cells from whole blood using density gradient centrifugation. See how to layer blood over the density medium and learn two different ways of harvesting cells from the blood plasma-density medium interface.</div></div>

[STEPS]
?. Dilute the blood sample at a 1:1 volume ratio with the appropriate medium.
?. Gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.
?. Centrifuge
?. Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface.
Alternatively: You can first remove the upper layer and then collect the cells.
?. Finally, wash the harvested cells twice in the appropriate buffer. The cells are now ready for EasySep™ cell separations or other downstream applications.
?. Add a volume of density gradient medium to a fresh tube according to the specifications for that density gradient medium.
TITLE: Tissue Harvesting | HuBMAP | JHU-TMC
AUTHORS: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
This protocol describes how to harvest human tissue biopsy and prepare it for histological processes

[STEPS]
SECTION: Prepare tissue collection container
1. For tissue collection container, we will prefill histology containers with 10% Neutral Buffered Formalin (NBF) 
SECTION: Prepare tissue collection container
2. Combine 100 mL of Formaldehyde (37% - 40%), 900mL of distilled water, 4g of Sodium dihydrogen phosphate monohydrate, and 6.5g of disodium hydrogen phosphate anhydrous.
SECTION: Biopsy - scalp
7. The surgical procedures for scalp skin include tissue expansion, nerve release,
hair transplantation, local tissue rearrangement.
SECTION: Prepare tissue collection container
3. Confirm that the pH of the solution is between 6.8 and 7.2.
SECTION: Prepare tissue collection container
4. Fill the solution into histology containers
SECTION: Prepare tissue collection container
5. Store at room temperature until use.
SECTION: Prepare tissue collection container
6. One can purchase this instead: VWR‱ Prefilled Histology Containers, 10% Neutral Buffered Formalin (NBF) Cat. No. 16004-121
SECTION: Biopsy - scalp
8. Hair bearing areas are chosen and areas with a paucity of hair follicles including
those from advanced androgenic alopecia or alopecia areata are excluded.
SECTION: Biopsy - trunk
9. We collect our samples from tissues normally discarded during surgical procedures.
The surgical procedures for trunk skin include deep inferior epigastric artery
perforator flaps, panniculectomy, abdominoplasty, reduction mammaplasty. 
SECTION: Fixation
10. Once the biopsy is harvested, we immediately put it into the tissue collection container to fix the tissue and avoid the degradation. The time delay between tissue excision and fixation, warm ischemic time, is a critical factor.

Note on the effect of delay to formalin fixation - Delay to
formalin fixation effect on breast biomarkers - PubMed (nih.gov)
SECTION: Fixation
11. Please refer to our tissue fixation protocol for the next steps
TITLE: Preparation of chemically competent E. coli cells
AUTHORS: Alice Pawlowski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Standard protocol to prepare cells for highly efficient DNA uptake using CaCl</span><span style = "vertical-align:sub;">2 </span><span> (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001). </span></div></div>

[STEPS]
?. Carefully suspend each pellet in 20 ml of icecold 100 m CaCl2 and incubate on ice for 1 h.
?. Inoculate a 5 ml overnight-culture in LB-medium without antibiotics.
?. Inoculate 100 ml LB-medium with 1/100 volume of the overnight culture and incubate to an OD600 = 0.5 - 0.6 at 37 °C and 230 rpm.
?. Transfer cells to two 50 ml sterile falcon tubes and incubate on ice for 20 min.From this step on, keep cells always cold.
?. Centrifuge for 5 min at 4500 rpm and 4 °C (pre-cool centrifuge) and discard the supernatant.
?. Carefully suspend each pellet in 2 ml icecold 85 mM CaCl2 containing 15% Glycerol.
?. Centrifuge for 5 min at 4500 rpm and 4 °C (pre-cool centrifuge) and discard the supernatant.
?. Pipette 100 to 200 µl aliquots in 1.5 ml tubes (work on ice) and store at -80 °C. (you can shock-freeze the cells in liquid nitrogen or in dry ice prior to storage at -80°C, but it is not required)For DNA transformation, thaw 100 µl of the competent cells on ice.
TITLE: Essential CBD Gummies Australia Price, Essential CBD Extract Gummies Review
AUTHORS: Essential CBD Gummies Australia Au

[DESCRIPTION]
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[STEPS]
TITLE: Improvements in episodic future thinking methodology:  Establishing a standardized episodic thinking control
AUTHORS: Kelseanna Hollis-Hansen, Sara E. O’Donnell, Jennifer S. Seidman, Spencer J. Brande, Leonard H. Epstein

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objective: </span><span>Delay discounting (DD) is the choice of a smaller immediate reward over a larger delayed reward, which has been associated with a number of maladaptive behaviors. Episodic future thinking (EFT), the ability to project oneself into the future, is an intervention designed to reduce DD. EFT has reliable effects on DD, but the size of the effect varies, which could be due in part to the use of different control groups. Episodic recent thinking (ERT) serves as a common control for many EFT studies, but the temporal window of “recent” cues ranges from 24 hours ago to 12 days past. Since prior research has shown that retrospection can lead to prospection, it may be important to ensure that EFT controls do not inadvertently lead to prospection for some participants thereby increasing the variability of the control condition. The present study sought to develop a comparison group that standardizes the time frame and experiences that are the basis for the recent thinking control.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods: </span><span>Participants (n=53, 18-45) were randomized to one of three conditions: EFT, ERT, or standardized episodic thinking (SET). Participants attended a laboratory appointment where they played mobile application games, created cues, and completed a DD task. </span></div><div class = "text-block"><span style = "font-weight:bold;">Results: </span><span>There was a significant difference between groups (</span><span style = "font-style:italic;">p</span><span><0.05), with EFT reducing discounting more than either control (p<0.05), and no differences between ERT and SET (p>0.05). </span></div><div class = "text-block"><span style = "font-weight:bold;">Conclusion: </span><span>This study established that SET provides an alternative control in EFT studies and provides the advantage of standardizing the participant’s recent experience without changing the relationship between EFT and recent thinking controls. </span></div></div>

[STEPS]