eamag/protocols-with-steps huggingface dataset:
TITLE: Aqueous (SBiP) Delipidation of a Whole Mouse Brain
AUTHORS: Andrew Recknagel, Kevin Cao, Naveen Ouellette, Molly Logsdon, Judith Baka, Jayaram Chandrashekar

[DESCRIPTION]
Aqueous strategies for whole-brain delipidation involve lipid removal via phase separation and detergent washes. SBiP is an aqueous biphasic buffer that extracts lipids at polar-nonpolar solvent interfaces. The brain is then washed with the detergent-based B1n buffer, which further disrupts residual membranes, forming micelles that can be washed out. When paired with organic delipidation, these steps can return a solvent-shrunken brain to normal size in phosphate buffer, suitable for post-delipidation antibody labeling. 

[BEFORE_START]
If performing this delipidation as part of the Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy protocol, first delipidate the whole mouse brain with organic solvents using the THF and DCM Delipidation of a Whole Mouse Brain protocol.  

[GUIDELINES]
It is recommended to etch identification information on the glass vial. Ink or label adhesive may dissolve if exposed to organic solvents.

[STEPS]
SECTION: SBiP Delipidation
2. Use a 20 mL vial for processing an adult mouse brain. All steps in this section are carried out on a carousel rotator  . 
Replace solution in vial with 20 mL SBiP for each of the following steps:
SBiP for 180 min 
SBiP for 360 min 
SBiP   
SBiP for 1440 min 
SBiP for 1440 min
SBiP for 1440 min 
SBiP for 1440 min 


 

 


SECTION: SBiP Delipidation
3. Wash the brain with 20 mL B1n buffer at Room temperature  .
SECTION: SBiP Delipidation
4. Replace B1n buffer to complete ~1440 min wash.
SECTION: SBiP Delipidation
5. Wash the brain with 1X PBS, rotating at Room temperature for the following steps:
PBS for 60 min 
PBS for 120 min 

SECTION: SBiP Delipidation
1. Delipidate with aqueous SBiP solution

Start with a whole mouse brain perfused with 4% PFA, post-fixed, stored in PBS in a 20 mL vial.  

SECTION: SBiP Delipidation
6. Aqueous (SBiP) delipidation complete. Store in 1X PBS 0.05% Azide for up to 6 months.
TITLE: Immunochemistry on paraffin sections
AUTHORS: miquel.vila

[DESCRIPTION]
Immunochemistry protocol on paraffin-embedded rat brain sections 

[STEPS]
SECTION: 1. Deparaffinization and hydratation :
1. Put the slides 30 min in the incubator at 60ºC.
SECTION: 1. Deparaffinization and hydratation :
2. Wash 3x3 min in Xilen.
SECTION: 1. Deparaffinization and hydratation :
3. Wash 1x10 min in ethanol 100%.
SECTION: 1. Deparaffinization and hydratation :
4. Wash 1x10 min in ethanol 95%.
SECTION: 1. Deparaffinization and hydratation :
5. Wash 1x5 min in ethanol 70%.
SECTION: 1. Deparaffinization and hydratation :
6. Wash 2x5 min in TBS 1X.
SECTION: 2. Blocking endogenous peroxidase:
7. Put the slides 10 min in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol
SECTION: Wahsing
8. Wash 3x5 min in buffer TBS 1X.
SECTION: Antigen retrieval
9. Put sections in 200 mL of citrate buffer 10mM, pH 6
SECTION: Antigen retrieval
9.1. Put sections in a boiling water bath for 20 min
SECTION: Antigen retrieval
10. Let sections cool down for 20 min at RT
SECTION: Washing
11. Wash 3x5 min in TBS 1X.
SECTION: Blocking
12. Put wet paper in a black box for immunohistochemistry
SECTION: Blocking
13. Gently dry and circle sections with the hydrophobic pen ImmEdge Vector H 4000 without touching the tissue 
SECTION: Blocking
14. Blocking in TBS 1X + 5% NGS (200uL/slides) 1h at RT
SECTION: 1ary antibody
15. Gently wipe off the water ofthe slides
SECTION: 1ary antibody
16. Put 200ul/slide of TBS 1X + 2% NGS + primary Ab48/72h (it depends of the Ab) at 4ºC (cold room)
SECTION: Washing
17. Wash 3x5min in TBS 1X Buffer.
SECTION: 2ary antibody
18. Gently wipe off the water ofthe slides
SECTION: 2ary antibody
19. Put 200ul/slide TBS 1X + 2% NGS + Secondary Ab 1h at RT.
SECTION: 2ary antibody
19.1. At this step it is important to prepare ABC solution and let it, at least, 30 min on the shaker
SECTION: Washing
20. 3x5min in TBS 1X 
SECTION: Peroxidase staining
21. Incubate 1 hour at RT with ABC solution (4drops A/B in 10 mL of TBS 1x)
SECTION: Washing
22. 3x5min in TBS 1X 
SECTION: Developing preparation
23. In aluminium foil: DAB Standard Kit: 1 drop of reagent B in 1 mL of reagent A (gives rise to brown staining)
SECTION: Developing preparation
23.1. In aluminium foil: Vector SG: 3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS (gives rise to blue staining)

SECTION: Developing
24. Cover tray plates with aluminium foil 
SECTION: Developing
24.1. Put 200 uL on each section and put a cardboard box on it to keep darkness for a time ranging from 3-15 minutes depending on the antibody used
SECTION: Developing
24.2. Remove with an air-pump equipment and clean the material with bleach
SECTION: Washing
25. 3x5min in TBS 1X 
SECTION: Dehydratation
26. 1 min in ethanol 70%
SECTION: Dehydratation
27. 1 min in ethanol 95%
SECTION: Dehydratation
28. 1 min in ethanol 100%.
SECTION: Dehydratation
29. 2x5 min in xylene
SECTION: Mounting
30. Puta line of mounting medium(DPX) byslide.Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles
31. Let dry the slides on the tray in the hood overnight
TITLE: NCEM Drop - Paramyxo confirmation, generation of RNP (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
Generate RNP to confirm Paramyxoviurs

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Conventional
4. Adsorb 10 µL sample to grid 10 min , inspect to ensure sample does not dry out.

SECTION: Conventional
5. Drain excess sample from grid using filter paper, leave wet.
SECTION: Conventional
6. Stain  1 min 

7. Drain & dry using filter paper
SECTION: Generation of RNP
2. Prepare 0.5 Mass / % volume  in TC Water.

SECTION: Generation of RNP
3. Mix 1:1 with the sample for 5 min
TITLE: Protocols for the draft genome assembly of the eastern banjo frog Limnodynastes dumerilii dumerilii
AUTHORS: Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang

[DESCRIPTION]
Background: Amphibian genomes are usually challenging to assemble due to large genome size and high repeat content. The Limnodynastidae is a family of frogs native to Australia, Tasmania and New Guinea. As an anuran lineage that successfully diversified on the Australian continent, it represents an important lineage in the amphibian tree of life but lacks reference genomes.

Find: Here we sequenced and annotated the genome of the eastern banjo frog Limnodynastes dumerilii dumerilii to fill this gap. The total length of the genome assembly is 2.38 Gb with a scaffold N50 of 285.9 kb. We identified 1.21 Gb of non-redundant sequences as repetitive element and annotated 24,548 protein-coding genes in the assembly. BUSCO assessment indicated that more than 94% of the expected vertebrate genes were present in the genome assembly and the gene set.

Conclusions: We anticipate that this annotated genome assembly will advance the future study of anuran phylogeny and amphibian genome evolution.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is aimed at amplifying the 18S rRNA hypervariable region 9 (18S V9) in eukaryotes with a focus on microbial eukaryotes. Amplicons generated using this protocol can then be sequenced using the Illumina platform. The primers (1391F, EukBr) utilized in this protocol are based on the primer utilized in Amaral-Zettler et al 2009 and the Earth Microbiome Project (EMP).
 
This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASAvdgdg), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.
 
Citations
Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes. PLoS ONE 4(7): e6372. doi:10.1371/journal.pone.0006372

Earth Microbiome Project - http://www.earthmicrobiome.org/emp-standard-protocols/18s/

[BEFORE_START]
Disinfect work surfaces with 10% bleach, followed gfvdfdsfdgfdgfdgdfgdfgfdgfby 70% ethanol, then RNase Away and pipets with RNase Away. UV pipets, molecular grade water, and tube racks for 20 minutes prior to starting protocol.

[GUIDELINES]
Local containment involves the employment of biological safety cabinets for initial specimen disinfection and DNA extraction. Such cabinets should not be used for PCR setup, as this procedure should be conducted apart from the aforementioned disinfection and extraction to minimize contamination.
 
Do not use any tube or plate that is not appropriate for the PCR machine you are using.
Make sure tubes and especially plates are well sealed before you begin run.
Clean up any spilled solutions and dispose of in appropriate biohazard boxes.
Be careful with PCR machine lids. These can bgdfe fif you slam or drop lids.
Make sure PCR heater block is clean before you start a run. Check each tube receptacle before you start.
Distribute tubes evenly across block so lid will seat flat against top of tubes for even heating and sealing.
Turn PCR machine off when you are done using.

[STEPS]
SECTION: PCR
?. 
SECTION: PCR
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SECTION: PCR
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SECTION: PCR
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SECTION: PCR
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SECTION: Quality control, PCR clean-up and sequencing parameters
?. 
SECTION: Quality control, PCR clean-up and sequencing parameters
?. 
SECTION: Quality control, PCR clean-up and sequencing parameters
?. 
SECTION: Quality control, PCR clean-up and sequencing parameters
?. 
SECTION: Quality control, PCR clean-up and sequencing parameters
?. 
SECTION: Sequencing
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SECTION: Sequencing
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SECTION: Sequencing
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SECTION: Sequencing
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SECTION: Sequencing
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SECTION: Sequencing
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>March, 2012; based on sample preparation guide v2, catalog # RS-930-1021, part # 15026495 Rev. A, August 2011</p>
<p> </p>
<p>Modifications: samples in PCR tubes (because a plate/seal will not be used, some numbered steps are skipped); include two rounds of poly-A selection (with two separate sets of beads); repeat clean-up step after PCR to remove additional adapters; elute with 1.5 ul less volume off of beads (to leave 1 ul behind, not 2.5 ul)</p>

[GUIDELINES]
<p><strong>PCR Programs</strong></p>
<p> </p>
<p><strong>mRNA Denaturation</strong>: 65ºC for 5 min, 4ºC hold</p>
<p><strong>mRNA Elution 1</strong>: 80ºC for 2 min, 25ºC hold</p>
<p><strong>80ºC</strong>: 80ºC forever (pre-heat, then run for 2 min)</p>
<p><strong>Elution 2 – Frag – Prime</strong>: 94ºC for 8 min, 4ºC hold</p>
<p><strong>1<sup>st</sup> Strand</strong>: 25ºC for 10 min, 42ºC for 50 min, 70ºC for 15 min, 4ºC hold</p>
<p><strong>2<sup>nd</sup> Strand</strong>: 16ºC forever (pre-heat, then run for 1 hour)</p>
<p><strong>End Repair</strong>: 30ºC forever (pre-heat, then run for 30 min)</p>
<p><strong>A Tail</strong>: 37ºC forever (pre-heat, then run for 30 min)</p>
<p><strong>Ligate Adapters</strong>: 30ºC forever (pre-heat, then run for 10 min)</p>
<p><strong>PCR</strong>: 98ºC for 30 sec; 15 cycles: 98ºC for 10 sec, 60ºC for 30 sec, 72ºC for 30 sec; 72ºC for 5 min, 10ºC hold</p>

[STEPS]
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TITLE: Pooling
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
The throughput of modern sequencers grows higher and higher, allowing hundreds of millions of reads in a single microfluidic chamber. For many purposes this is actually far more than a single library requires. To keep sequencing cost-effective, researchers often pool together multiple libraries before sequencing, each with a unique molecular barcode (or unique combination of multiple barcodes). The sequencer then reads each library molecule's biological base sequence as well as the barcode sequence; these barcodes are matched back to the sequences expected from the libraries, and thus each molecule can be attributed to its library of origin even though the libraries were mixed. The pool of libraries needs to be prepared at a specific molarity. Thus when you combine libraries into a pool to sequence together, you must take care to match not only their relative molarities but also the absolute total molarity of the pool.

[STEPS]
SECTION: Preparations
2. DNA Away and Ethanol the workstation.
SECTION: Preparations
3. Remove PCR plate(s) from the-20 °C freezer and thaw plates to be pooled while finishing workstation prep.

SECTION: Preparations
1. Print plate table with volume of each amplicon to be pooled together (equal molar).
SECTION: Preparations
4. Spin thawed plates down in centrifuge.
SECTION: Pooling Sample(s)
5.1. Use a temporary seal to help keep wells uncontaminated and to keep track of samples being added.
SECTION: Pooling Sample(s)
5. Pipette the correct volume from the PCR plate according to the table into the labeled 1.5 mL tube 
SECTION: Pooling Sample(s)
5.2. Clearly mark off when a sample has been added to the pooling tube.
SECTION: Pooling Sample(s)
6. Once an entire plate’s samples have been added, place a freezer seal on the plate and seal well.
SECTION: Pooling Sample(s)
7. When all samples have been added to the pooling 1.5 mL tube, clean the workstation and place the PCR plates and pooling tube in the -20 °C freezer for temporary storage.
TITLE: Background control of Gotcha RCA
AUTHORS: Chia-Hsien  Shih

[DESCRIPTION]
This protocol aims to verify that GotCha design works as expected.



[STEPS]
SECTION: Preparation
1. Add 5 µL of functional beads(Gotcha) into eppendorf

SECTION: Preparation
2. Centrifuge for 15000 rpm, 5 min and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.

SECTION: Protocol of Control group without miRNA
3. Add 3 µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads

SECTION: Protocol of Control group without miRNA
4. Add 23.4 µL RNase-free water

SECTION: Protocol of Control group without miRNA
5. Add 3 µL of 2mM dNTPs

SECTION: Protocol of Control group without miRNA
6. Add 0.6 µL of 10U/ul phi29 polymerase

SECTION: Protocol of Control group without miRNA
7. Pipetting to mix well


SECTION: Protocol of Control group without miRNA
8. Incubate for 120 min at Room temperature

SECTION: Protocol of Control group without miRNA
9. Add 1.5 µL of 20X evagreen dye 

SECTION: Protocol of Control group without miRNA
10. Add 1.5 µL of 0.5M EDTA buffer to suspend the reaction

SECTION: Protocol of Control group without phi29 polymerase
11. Add 3 µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads

SECTION: Protocol of Control group without phi29 polymerase
12. Add 21 µL RNase-free water


SECTION: Protocol of Control group without phi29 polymerase
13. Add 3 µL of 2mM dNTPs


SECTION: Protocol of Control group without phi29 polymerase
14. Add 3 µL of 100nM miRNA

SECTION: Protocol of Control group without phi29 polymerase
15. Pipetting to mix well


SECTION: Protocol of Control group without phi29 polymerase
16. Add 1.5 µL of 20X evagreen dye 

SECTION: Protocol of Control group without phi29 polymerase
17. Add 1.5 µL of 0.5M EDTA buffer to suspend the reaction

SECTION: Measuring
18. Load 20 µL of reaction solution into 384-well plate

SECTION: Measuring
19. Measure the fluorescence excitation and emission intensity
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
BONCAT is a technique that allows for the visualization and isolation of metabolically active cells from complex environmental enrichments. This is achieved by incubating a sample with synthetic methionine analogs AHA (l-azidohomoalanine) or HPG (l-homopropargylglycine). Instead of the sulfhydryl side group of methionine , these analogs contain an azide or alkyne functional group, respectively, which can be chemically tagged with a fluorophore using a process termed “click-chemistry”. Throughout the incubation period actively growing cells incorporate these synthetic amino acids into newly synthesized proteins. After harvesting cells from a live enrichment, the cells are tagged with the fluorophore. This allows for microscopic visualization along with FACS-based isolation of the metabolically active cells in a community. Downstream analysis can include single-cell sequencing or metagenomics techniques to determine taxonomy and functions of isolated organisms. 
 
 
Notes:
 
 Working with low-biomass samples has an inherent high-risk of contamination. Be mindful of sterility at all times. Perform all work in the biological safety cabinet and make sure to sterilize the hood with UV prior beginning work
 Some of the BONCAT reagents are light-sensitive, be careful to pay attention to if a particular step in the protocol needs to be performed in the dark
The click-chemictry reaction is very sensitive to oxygen, so take care not to vortex/ introdroduce air unnecessarily  
 Try to plan when you harvest incubations so that you are certain you will be able to perform the click-chemistry process the next morning

[GUIDELINES]
Notes:
 
 Working with low-biomass samples has an inherent high-risk of contamination. Be mindful of sterility at all times. Perform all work in the biological safety cabinet and make sure to sterilize the hood with UV prior beginning work
 Some of the BONCAT reagents are light-sensitive, be careful to pay attention to if a particular step in the protocol needs to be performed in the dark
The click-chemictry reaction is very sensitive to oxygen, so take care not to vortex/ introdroduce air unnecessarily  
 Try to plan when you harvest incubations so that you are certain you will be able to perform the click-chemistry process the next morning

[STEPS]
SECTION: Incubation Set Up (Time required: ~ 1.5 – 2.0 Hours)
?. 
SECTION: getting things prepped pt. 2
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SECTION: getting things prepped pt. 3
?. 
SECTION: weigh rock samples
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SECTION: Sterilize outside of rocks ( This may not be applicable to your experimental design!)
?. 
SECTION: Crush it.
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SECTION: Crush it pt. 2
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SECTION: Weigh crushed rock
?. 
SECTION: Add media and non-canonical amino acid
?. 
SECTION: And wait.
?.
TITLE: Protocol for mixed cortical striatal cell culture 
AUTHORS: Chuyu Chen, Ciarra Smith, Loukia Parisiadou

[DESCRIPTION]
This protocol describes a optimised protocol for culturing cortical and striatal neurons from postnatal pup brains

[STEPS]
1. Add 500µl of poly-D-lysine solution to each well that will be used for plating
2. Allow to sit inside the hood for at least 30mins (this plate will stay under the hood until dissection is finished)
3. Set BME + supplement and BME+AraC serum in water bath
4. Add 1-2ml of BMS alone into 2 wells of a 24-well plate (not coated)
5. Add 4-5ml of BMS alone into 16mm culture dish
6. Place dish on ice and leave BME inside the hood
7. Dissect P0/P1 brains on ice in to dissection medium
8. Aspirate medium gently, add 1ml of enzyme and incubate for 20 minutes at 37 degrees
9. Note: It is better to cut tissue sample using scalpel to allow more area for optimal enzymatic digestion
10. Wash 3X with 1ml of room temp BME medium without supplement to wash out the enzyme
11. Add 2ml of medium to well
12. Gently pipette 15 times and transfer to 15ml tube
13. Wait ~1min for large chunks to settle
14. Carefully remove supernatant and transfer to fresh 15ml tube
15. Centrifuge: 300g, 4°C, 4min
16. Remove media and resuspend in 1ml of media + serum
17. Remove poly-d-lysine from 24-well dishes
18. Add 500µl of medium + serum to each well
19. Count cells: 5x10^5 cells/well
20. Plate 1:2 striatum:cortex
21. Incubate for 1hr then change media
22. Add GDNF to BME-(without serum; 1µl of GDNF in 2.5ml)
23. Incubate cells in a CO2 incubator
TITLE: Explant Surgery: Chronic recoverable Neuropixels in mice
AUTHORS: Emily A Aery Jones

[DESCRIPTION]
This protocol collection explains how to build a low-cost, lightweight system to implant 1 Neuropixels 1.0 probe or 2 Neuropixels 2.0 probes into mice, record during freely moving behavior, then recover the probes for future use. This protocol explains how to recover a previously implanted Neuropixel probe and clean for future use. See full collection for more details.

[STEPS]
SECTION: Prepare mouse
1. Set O2 flow rate to 1-1.5L/min and isoflurane to 3%. Place mouse in anesthetic chamber.
 

SECTION: Prepare mouse
2. When breathing has slowed to 1Hz, move animal to the toothbar. Switch isoflurane from chamber to nosecone. Wait until unresponsive to pedal reflex test, then lower to 1.5% isoflurane.
 



SECTION: Prepare mouse
3. Set the nosecone to the position you recorded from the implant surgery. Attach mouse to headbar stereotactic adapters.
SECTION: Remove probe
4. Remove all the tape to expose the screws and probe.
SECTION: Remove probe
5. Cut the ground wire above the pin.
SECTION: Remove probe
6. Attach the Sensapex holder and set the stereotax to the angle used in the implant surgery. Tax to the dovetail cap. A fully open Sensapex holder can be taxed in from anterior or dorsal to the dovetail holder.
SECTION: Remove probe
7. Adjust the screws so the holder grips the dovetail cap.
SECTION: Remove probe
8. Unscrew the wings from the body. Screws can be saved for future surgeries.
SECTION: Remove probe
9. Slowly tax up to bring the probe & body piece away from the animal. Set back to 0 degrees and adjust the arm so the probe is away from the surgical field.
SECTION: Remove probe
10. Lower the probe into tergazyme and let sit for 30 minutes, then rinse with dH2O.
SECTION: Perfuse
11. Remove the mouse from the stereotax and prepare for perfusion.
  

SECTION: Re-use probe
12. Assemble following the Assembly protocol, skipping the steps that modify the probe (sharpening, soldering wire to the PCB, attaching the body piece). Extend the ground wire back to full length and attach a new pin.
TITLE: DNA Quantification
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for DNA quantification. To see the full abstract and additional resources, please visit </span><a href="https://www.addgene.org/protocols/dna-quantification" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/dna-quantification/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Before measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, but with no DNA added. 'Blanking' measures the background inherent to the machine and your solvent.
?. If using a NanoDrop to measure your samples, place - of mini-prepped DNA onto the pedestal.
1 µl
2 µl
?. Close the lid and click measure, be sure to record the concentration and purity.
Note, purity is measured under the 260/280 column (A good purity ranges from 1.80-2.00).
?. Repeat for each sample.
Notes: Keep in mind that despite the accuracy of the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible that the difference between them has affected the accuracy of the NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary significantly in concentration.DNA dissolved in water is going to have a greater variance in concentration readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and consistent readings from DNA in a buffered solution.