eamag/protocols-with-steps huggingface dataset:
TITLE: Adverse Event Reporting (Part 7 of &#34;Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain&#34;)
AUTHORS: Yiting Lin

[DESCRIPTION]
This is Part 7 of "Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain".
Physical activities have little risk of heart attack and other life-threatening conditions. During the test and trial, if there are adverse reactions such as acute aggravation of neck pain, the test or intervention will be stopped immediately, and emergency treatment will be conducted.

[GUIDELINES]
Adverse Event Reporting:

In this study, the tests and interventions may include physical activities, which may irritate neck pain. Physical activities have little risk of heart attack and other life-threatening conditions. During the test and trial, if there are adverse reactions such as acute aggravation of neck pain, the test or intervention will be stopped immediately, and emergency treatment will be conducted. If there is any emergency, the principal investigator(s) will provide basic life support including chest compressions and ventilation until emergency medical staff are on hand. Participants can consult the clinicians if they are experiencing any side effects from taking part in the study and should also inform the principal investigator.

[STEPS]
TITLE: Immunofluorescence
AUTHORS: Daehun Park, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the immunofluorescence staining of neurons.

[STEPS]
SECTION: Protocol
1. Wash cultured hippocampal neurons with pre-warmed tyrode.
SECTION: Protocol
2. Fix the cells with fixative solution for 15 min at Room temperature.

SECTION: Protocol
3. After fixation, wash the cells with PBS. 
SECTION: Protocol
5. Wash the cells briefly with PBS and incubate the cells with primary antibodies (1:500 ~ 1:2000) in a blocking buffer for 60 min at Room temperature on a rocking platform.

SECTION: Protocol
6. Wash with PBS on a rocking platform.
SECTION: Protocol
7. Incubate the cells with secondary antibodies (for example Alexa-fluor-labeled) (1:1000) in a blocking buffer for 45 min on a rocking platform atRoom temperature in the dark.

SECTION: Protocol
8. Decant the secondary antibodies and wash with PBS.
SECTION: Protocol
9. Observe the fluorescence signal using an inverted confocal microscope or mount the samples with Prolong Gold antifade reagent for long-term storage
SECTION: Protocol
1.1. Wash cultured hippocampal neurons with pre-warmed tyrode. (1/3)
SECTION: Protocol
1.2. Wash cultured hippocampal neurons with pre-warmed tyrode. (2/3)
SECTION: Protocol
1.3. Wash cultured hippocampal neurons with pre-warmed tyrode. (3/3)
SECTION: Protocol
4. Incubate with blocking buffer for 30 min atRoom temperature.

SECTION: Protocol
3.1. Wash the cells with PBS. (1/3)
SECTION: Protocol
3.2. Wash the cells with PBS. (2/3)
SECTION: Protocol
3.3. Wash the cells with PBS. (3/3)
SECTION: Protocol
6.1. Wash with PBS for 2 min on a rocking platform. (1/5)

SECTION: Protocol
6.2. Wash with PBS for 2 min on a rocking platform. (2/5)
SECTION: Protocol
6.3. Wash with PBS for 2 min on a rocking platform. (3/5)
SECTION: Protocol
6.4. Wash with PBS for 2 min on a rocking platform. (4/5)
SECTION: Protocol
6.5. Wash with PBS for 2 min on a rocking platform. (5/5)
SECTION: Protocol
8.1. Wash with PBS for 2 min each in the dark. (1/5)

SECTION: Protocol
8.2. Wash with PBS for 2 min each in the dark. (2/5)
SECTION: Protocol
8.3. Wash with PBS for 2 min each in the dark. (3/5)
SECTION: Protocol
8.4. Wash with PBS for 2 min each in the dark. (4/5)
SECTION: Protocol
8.5. Wash with PBS for 2 min each in the dark. (5/5)
TITLE: LEGACY01: QUALITY ASSURANCE AND CONTROL
AUTHORS: Katrina M Pollock, Calliope Dendrou

[DESCRIPTION]
This protocol details quality assurance and control in an experimental medicine study of seasonal influenza vaccination responses in Lymph nodE single-cell Genomics in AnCestrY (LEGACY01).

[GUIDELINES]
QUALITY ASSURANCE AND CONTROL

Quality assurance (QA) includes all the planned and systematic actions established to ensure the study is performed and data generated, recorded and reported in compliance with the principles of GCP and any regulatory requirements. Quality control (QC) includes the operations to ensure that requirements for the study activities are fulfilled.

SAFETY AND RIGHTS OF PARTICIPANTS

Participants will be donating tissue and undergoing immunisation using procedures that are commonly undertaken for patients in the NHS. Confidential information will be kept allowing safety assessments to be made throughout the study.

PROJECT DESIGN AND RELIABILITY

A feasibility study is already underway lead by the Chief Investigator (EAVI2020_01 FNA sub study). This study has indicated that lymph node sampling is feasible, safe and with ample yields to be undertaken as part of human vaccine research.

CLINICAL DATA RECORDING AND MONITORING

Worksheets for the study will be created for each study visit. Completed worksheets will undergo regular QC assessments by the study clinical team. Data will be stored by means of an electronic database using REDCap or similar software system.

AUDITS

The study may be subject to audit by Imperial College London, under its remit as sponsor, and other regulatory bodies to ensure adherence to GCP and the UK Policy Framework for Health and Social Care Research.

REFERENCE

Day S, Kaur C, Cheeseman HM, de Groot E, McFarlane LR, Tanaka M, Coelho S, Cole T, Lemm N-M, Lim A, Sanders RW, Asquith B, Shattock RJ and Pollock KM (2022) Comparison of blood and lymph node cells after intramuscular injection with HIV envelope immunogens. Front. Immunol. 13:991509. doi: 10.3389/fimmu.2022.991509

[STEPS]
TITLE: Calculation_and_Normalization_of_Relative_Gene_Abundance
AUTHORS: Fan Wei, Fan Jiang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Calculate the relative abundance of gene for metagenome analysis.</div></div>

[STEPS]
?. Mapping the clean reads (abc.clean.read1.fq.gz, abc.clean.read2.fq.gz) of each sample  to all sequences of the indexed gene catalog
?. 
?. Construction of the bwa index for the gene catalog (MGCA_gene_Catalog.fasta)
?. Selecting the first and last columns from the output file (abc.clean.fq.gz.sam.gz..mapping.abundance), which representing the gene id and the normalized relative abundance of the gene
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for extraction of tissue RNA by Qiagen Mini Kit.</p>

[BEFORE_START]
<p>Clean the benches and all the material that you will use with alcohol 70.<br />Use tips with filter.</p>
<p> </p>
<p>Add 4 volumes of ethanol 100 to 4 volume RPE buffer.</p>
<p>To lyse your sample, work with β-mercaptoethanol or 2 M dithiothreitol.<br />For β-mercaptoethanol, use 10 μl for every 1 ml of the RLT buffer.<br />For 2 M dithiothreitol, use 20 μl for every 1 ml of the RLT buffer.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: PCR Clean-up
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to isolate the known DNA fragments from the PCR product without running the gel electrophoresis. 

[STEPS]
1. Add 5 times the sample's volume of   into the PCR tube with the PCR product.

3. Centrifuge the tube at 13 rpm, 1 min.

4. Discard the flow-through and place the QIAquick column back into the same tube.
5. Add 700 µL of   into the QIAquick column.

7. Discard the flow-through and place the QIAquick column back into the same tube.
11. Centrifuge the emptied QIAquick column at 13 rpm, 1 min to remove residual  .

12. Transfer the QIAquick column into the newly labelled Eppendorf tube.
13. Add 30 µL of DI water into the QIAquick column.

14. Centrifuge it at 13 rpm, 1 min, ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.
15. Discard the QIAquick column, the solution left in the Eppendorf tube contains the DNA fragments.
16. Use the Nanodrop to measure and record the purity and concentration of the DNA fragments.
 

2. Transfer the whole solution into a QIAquick Spin Column (purple tube) and ensure that the sample is dripped onto the white membrane in the column.
6. Centrifuge it at 13 rpm, 1 min.

8. Add 700 µL of   again into the QIAquick column.

9. Centrifuge it at 13 rpm, 1 min.

10. Discard the flow-through and place the QIAquick column back into the same tube.
TITLE: Isolation of NeuN+ cells from brain tissue (for CUT and RUN)
AUTHORS: anita.adami

[DESCRIPTION]
This protocol describes the steps to isolate NeuN+ cells from brain tissue in preparation for CUT and RUN

[STEPS]
SECTION: Isolation of NeuN+ cells
1. Nuclei were isolated from frozen tissue as described here. 
SECTION: Isolation of NeuN+ cells
2. Before FACSing, nuclei were incubated with Recombinant Alexa Fluor® 488 Anti-NeuN antibody [EPR12763] - Neuronal Marker (Abcam, Cat# ab190195, RRID:AB_2716282) at a concentration of 1:500 for 30 min on ice as according to Spalding, K.L. et al. (2013).

SECTION: Isolation of NeuN+ cells
3. The nuclei were run through the FACS at 4 °C with a low flow rate using a 100 mm nozzle and 300.000 nuclei Alexa Fluor – 488 positive nuclei were sorted. 

SECTION: Isolation of NeuN+ cells
4. The sorted nuclei were pelleted at 1,300 x g for 15 min and resuspended in 1 mL of ice-cold nuclear wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete protease inhibitors, 0.1% BSA) and 10 µL per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (Pipette tips for transferring nuclei were pre-coated with 1% BSA), to perform CUT&RUN.
TITLE: Population Structure and Phylogenetics
AUTHORS: Graham Etherington

[DESCRIPTION]
The European polecat (Mustela putorius) is a mammalian predator which breeds across much of Europe east to central Asia. In Great Britain, following years of persecution the European polecat has recently undergone a population increase due to legal protection and its range now overlaps that of feral domestic ferrets (Mustela putorius furo). During this range expansion, European polecats hybridised with feral domestic ferrets producing viable offspring. Here we carry out population-level whole genome sequencing on domestic ferrets, British European polecats, and European polecats from the European mainland and find high degrees of genome introgression in British polecats outside their previous stronghold, even in those individuals phenotyped as ‘pure’ polecats. 

[STEPS]
SECTION: Structure analysis
3. 
To create ADMIXTURE plots for our data, we used the following pipeline:
BCFTOOLS prune for linkage, reformat the into Plink format, run Structure over a sensible range of K, chooseK and plot with Structure


SECTION: Structure analysis
3.1. Take the linkage pruned dataset from 'Phylogenetics, Step 3.2, and handle missing genotypes

 

SECTION: Structure analysis
3.2. Then run on k=2-5

 
To identify the best value of K, I used the --cv for bootstrap and CV estimates
SECTION: Structure analysis
3.3. grep out the CV errors to find the best (highest) value of K

 

SECTION: Structure analysis
3.4. Plot all of the Admixture plots together.
 

SECTION: Structure analysis
1. RAxML phylogeny


SECTION: Structure analysis
1.1. Run RAxML in two stages. The first is the 'check' and 'parse'  options that check the alignment and creates an *raxml.rba binary  alignment file. It also estimates the resources needed to run RAxML efficiently (cores and memory)

 


SECTION: Structure analysis
1.2. Using the resources advised by the previous step, run final step to produce the phylogeny.

 

SECTION: Structure analysis
2. Treemix phylogeny
SECTION: Structure analysis
2.1. The first step is to create a 'clust' file. The clust file contains three columns, where the first and second column both indicate the name of the individual and the third  column indicates the taxon name. This can be achieved by using a combination of bcftools and awk.

 

SECTION: Structure analysis
2.2. Next, we need as input a VCF file that has been pruned for linkage- disequilibrium (LD).
The input for this is the 'all_samples_genotyped_snps.vcf' file produced here:
https://www.protocols.io/view/gatk-nuclear-variant-discovery-and-consensus-assem-bqzgmx3w?step=2.2

We filtered for an LD of 0.8 over windows of 5Kb

 

SECTION: Structure analysis
2.3. Remove any missing data

 

SECTION: Structure analysis
2.4. Convert the VCF file into TreeMix format
The files required can be found here:
https://bitbucket.org/nygcresearch/treemix/downloads/plink2treemix.py
https://github.com/speciationgenomics/scripts/blob/master/vcf2treemix.sh


 

SECTION: Structure analysis
2.5. Then run TreeMix with 1 to 5 migration edges (-m parameter)
TITLE: U Mass - Protein metabolism
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity.  The hyperinsulinemic-euglycemic clamp is widely used in clinics and laboratories to measure insulin action on glucose utilization in humans and animals for clinical and basic science research.  Incorporation of radioactive-labeled glucose during hyperinsulinemic-euglycemic clamps makes it possible to measure glucose metabolism in individual organs in awake mice.  Impaired insulin sensitivity (insulin resistance) is a major characteristic of obesity and an early requisite event in the development of type 2 diabetes.</div></div>

[STEPS]
?. Mice are fasted overnight (~15 hours) prior to the start of experiment.
?. Chronic indwelling catheter is placed 5~6 days prior to experiment for intravenous infusion. (methods can be referred to M1023: Surgery-jugular vein cannulation)
?. Place a mouse in a rat-size restrainer with its tail tape-tethered at one end.
?. Expose and flush the intravenous catheter using saline solution. Then, connect the catheter to the CMA Microdialysis infusion pump.
?. Collect plasma sample (20 µl) before the start of infusion (basal-0 min) to measure basal glucose and insulin levels.
?. Start infusion of 20% dextrose to quickly reach a target hyperglycemia (~300 mg/dl glucose level) and maintain hyperglycemia by adjusting glucose infusion rates.
?. Collect plasma samples (10 µl each) at 10, 20, 30, 45, 60, 90, and 120 min to measure glucose levels. Adjust glucose infusion rates based on instantaneous glucose levels to maintain at target hyperglycemia.
?. Collect additional plasma samples (10 µl each) at 10, 20, 30, 45, 60, 90, and 120 min to measure insulin concentrations.
?. At the end of experiment, mice are euthanized, and pancreas may be collected for further studies.
?. For data analysis, plasma insulin concentrations may be plotted during the 120-min hyperglycemic clamp experiment, and area-under-curve may be calculated. Area-undercurve of insulin levels during hyperglycemic clamps may be directly correlated with insulin secretion and pancreatic β-cell function assuming there are no effects on insulin clearance rates.
?. Additional plasma samples may be collected to measure serum c-peptide concentrations which may further reflect glucose-induced insulin secretion and pancreatic β-cell function in awake mice.
TITLE: Transfection with PEI 
AUTHORS: JCPrice 

[STEPS]
?. Cells should be 50-70% confluency and have a healthy morphology
?. In a clean biosafety cabinet, remove serum media from cells (DO NOT TRYPSINIZE!)
?. Add 1.5 mL of serum-free cell culture media (DMEM or similar)
?. In a sterile tube dilute total plasmid DNA (µg) in serum-free DMEM. 6 well plate: 200 µl + 3 µg of total DNA per well; 24 well plate: 30 µl + 0.5 µg of total DNA per well Add PEI (1 µg/µL) to the diluted DNA. Mix immediately by vortexing or pipetting. The volume of PEI used is based on a 3:1 ratio of PEI (µg):total DNA (µg). This ratio can be optimized to maximize transfection efficiency.     a.	6-well plate: 9 µl of PEI(1 µg/µl) = 9 µg PEI to 3 µg DNA     b.	You will need 200 µL serum-free DMEM + 3 µgDNA+9 µg PEI for each well of a 6-well plate you plan to transfect.    c.	24 well plate: 1.5 µl of PEI(1 µg/µl) = 1.5 µg PEI to 0.5 µg DNA     d.	You will need 30 µl serum-free DMEM + 0.5 µg DNA + 1.5 µg PEI for each well of a 24-well plate.
?. For the negative control, add 9 µg PEI to 200 µL serum-free media - no plasmid DNA.
?. Incubate 15 minutes at r.t.
?. Add DNA/PEI mixture to cells using the volume per well described above
?. Allow cells to incubate with DNA/PEI mixture for 4 hours in the 37°C cell culture incubator.
?. After 4 hours, remove serum-free medium with DNA/PEI mixture and replace with 2-3 mL complete serum media.
?. Incubate cells for 24-48 hours, at 37°C to allow for plasmid expression and protein translation
?. Fix or harvest for downstream analysis.
TITLE: HEK3/LINC01509 Library Preparation
AUTHORS: Mathew Chu

[DESCRIPTION]
Library preparation for HEK3 recording site (LINC01509) using PCR and digestion.

[STEPS]
SECTION: HEK293T Cell Lysis
1. Make up 1:50 Thermolabile Proteinase K (NEB) in Viagen (cell).
2. Resuspend cells in 25 µL of Viagen/Proteinase K. 

Quickly transfer cells into a PCR strip to avoid viscosity.
3. Perform lysis in a PCR machine:

37 °C 60 min 
55 °C 30 min 
on ice 

Store lysates at -20 °C in the pre-PCR freezer.

SECTION: PCR Amplification of Genomic DNA
4. Make up a 100 µL PCR reaction per sample (Q5) using 10 µL of cell lysate.

SECTION: SPRI Bead Concentration
5. Purify amplicons by 1X SPRI bead cleanup, eluting in 10 µL of water.

SECTION: Digestion of Zero-Length Recordings
6. Digest zero-length recordings with 5 µL BccI in 50 uL volume:

37 °C 60 min 
65 °C 20 min 
on ice 

SECTION: Gel Separation and Extraction of Recordings
7. Perform gel electrophoresis in 4% LMP agarose at 3 V/cm for 60 min.

SECTION: Indexing PCR of Recombinant DNA
9. Using 1 µL of gel product, perform indexing PCR in 12 µL volume with Nextera primers.

SECTION: Gel Separation and Extraction of Recordings
8. Extract the region from 300 bp to 500 bp and elute in 12 µL of water.

Store gel products at -20 °C in the post-PCR freezer.

SECTION: SPRI Bead Purification
10. Purify amplicons by 1X SPRI bead cleanup, eluting in 10 µL of water.

SECTION: D1000 Tapescreen
11. Take 1 µL of each indexed amplicon to D1000 Tapescreen.

 

SECTION: PCR Amplification of Genomic DNA
4.1. 98 °C 30 s  
SECTION: D1000 Tapescreen
12. Pool indexed amplicons according to desired relative molarities for sequencing.


SECTION: PCR Amplification of Genomic DNA
4.2. 98 °C 5 s 
68 °C 10 s 
72 °C 20 s
SECTION: PCR Amplification of Genomic DNA
4.4. 72 °C 2 min 

SECTION: PCR Amplification of Genomic DNA
4.3.   

SECTION: Indexing PCR of Recombinant DNA
9.1. 98 °C 30 s 
SECTION: Indexing PCR of Recombinant DNA
9.2. 98 °C 5 s 
67 °C 10 s 
72 °C 20 s 
SECTION: Indexing PCR of Recombinant DNA
9.3.   
SECTION: Indexing PCR of Recombinant DNA
9.4. 72 °C 2 min
TITLE: Rotarod (40 RPM and 20 RPM with docking) 
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
This protocol describes motor function testing in mice using the rotarod by the Svenningsson lab.

Two options for the protocol are included: 1) 40 RPM constant speed after habituation and 2) 20 RPM with docking. 

[STEPS]
3. Using a paper tube, transfer each mouse of the same cage onto the rotarod (Ugo Basile, 47650). Always maintain order, mice one into line one, mice 2 into line 2 etc…
5. Lift the collecting platform and set up the protocol: either max speed of constant 40 RPM or 20 RPM docking. 
7. The time to fall is defined as the moment the mice fall in the receptacle or the moment the mice turn around the rotor more than 5 consecutive times. The maximum trial length is 2 min.
9. Analyze for each mouse, the time to fall for each session, the average and and max time.
6. Following habituation, start the rotarod program to reach a speed of 40 RPM within the first 5s of the protocol.
6. Following habituation, start the rotarod program to reach 20RPM within the first 5 s and proceed for 5 rotations. Then the wheel will stop for 1s and turn towards the opposite direction at 20 RPM and proceed to 5 rotations and repeat that for a maximum of 120 s.
7. The time to fall is defined as the moment the mice fall in the receptacle or the moment the mice turn around the rotor more than 5 consecutive times. The maximum trial length is 2 min.
8. The apparatus was thus cleaned thoroughly between each group with 50% ethanol followed by water.
9. Repeat the test 3 times with at least 6 hr between each trial.
1. Transfer the cage into the experimental room at least 1 hr before testing.
2. To avoid stress, always put the cage into a ventilated cage rack.
4. Wait 2-3 minutes for the mice to habituate to the apparatus.
8. Repeat the experiment 3 times with at least 6 hr between each trial. If an animal does not fall or turn more than 5 times within 120s of the trial, stop the trial and record “120” as the individual time.
10. In the 20 RPM docking protocol, most of the animals will
fall around 20 to 25s of the first session that corresponds to the first change
of direction. After repeating the protocol (session 2-3), wild type mice should
be able to maintain for longer period and turn their body at each revolution.
11. The time to fall is defined as the moment the mice fall in the receptacle or the moment the mice turn around the rotor more than 5 consecutive times. The maximum trial length is 2 min.
TITLE: mPSM protocol
AUTHORS: Takehito  Tomita

[DESCRIPTION]
Protocol to perform 2D ex vivo segmentation assay (Lauschke et al. 2013).

[STEPS]
1. Coat dishes/plates with fibronectin.
1.1. Dilute fibronectin (Sigma, Cat.No: F1141) with PBS to 50ug/mL (20 fold dilution).
1.2. Fill each well of 8-well plate (NuncTMLab-TekTMII Chambered Coverglass, Cat.No: 155409PK) with ~160μl of fibronectin solution and incubate for 1 hour in 37°C.
1.3. Remove all fibronectin solution from the well(s) and dry for 1 hour at room temperature (~25°C).
2. Prepare dissection medium and culture medium.
2.1. Mix the following ingredients in a 50 mL falcon tube:

0.4 g 
400 µL 
35.52 µL 
40 mL 
Vortex well.
2.2. Separate 10 mL from the mix in step 2.1 and filter through a 0.22μm filter (Millex-GV, Cat.No: SLGV033RS) into a fresh 15 mL falcon tube. Add 100 µL to achieve  
100U/mL. This mix is used as culture medium. Equilibrate in 37°C 5% CO2 until use.
2.3. Add 510 µL to the remaining 30 mL mix in step 2.1. This mix is used as dissection medium. It can be kept at the bench until use.

3. Wash the dried wells with ~200μL culture medium per well. Apply fresh culture medium (160uL ~ 300μL per well) and equilibrate in 37°C 5% CO2 until use.
4. 
Extract mouse embryos and collect tails in dissection medium.


5. Cut the tail bud using a scalpel in dissection medium. Be careful not to flick the tail buds as this damages the tissue.
6. Wash the tail buds once in a dish containing culture medium. Quickly move the tail buds to the fibronectin coated wells. Align the tissue orientation using forceps to place the cut side downward.
7. Incubate in 37°C 5% CO2 for 1hr to let the tissue attach.


8. Place the well into the metal box to fit into the microscope. Add a strip of wet paper towel inside the metal box to prevent drying. Carefully move the box into the microscope, as tissue can detach quite easily.
9. Set up imaging.