eamag/protocols-with-steps huggingface dataset:
TITLE: Nuclei isolation from fresh and frozen brain tissue - for single nucleus RNAseq or 10x Multiome
AUTHORS: Isabelle Schmutz, Jade Carter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol is for nuclei isolation from frozen mouse brain or human postmortem brain and processing of these nuclei for loading on single cell sequencing platforms. Nuclei are prepared by dounce homogenization under mild cell lysis conditions. They are purified from myelin, subcellular debris and ambient RNA using centrifugation through an iodixanol cushion. Isolated nuclei can be directly loaded onto a single nucleus sequencing platform or they can be additionally permeabilized for combined single nucleus RNAseq/ATACseq. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The protocol is compatible with working on small amounts of tissue, using microtubes and benchtop centrifuges. We have successfully prepared nuclei from as little as 10 mg of frozen mouse brain tissue and processed them through the 10x Genomics Multiome assay.</div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
TITLE: Protocol 4: Creating depressions in induction media plates
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
Transformation is carried out by co-culturing Agro and Spizellomyces on induction media (“IM”). Once its virulence genes are induced, Agro will infect Spizellomyces and the plasmid the transfer DAN is randomly incorporated into the Spizellomyces genome. The IM media and agar plates (see recipes) should be no more than 1 month old, as the hormone Acetosyringone is likely to degrade, decreasing its effectiveness. For each plasmid, four ratios of Agro and Sp will be made and plated onto one plate (See Protocol 5.3). Prepare IM plates with small depressions using a glass culture tube to keep the co-culture liquids from touching each other.

[BEFORE_START]
The protocol below outlines how to prepare the depressions and should be practiced until the agar is no longer scarred. Prepare these plates within a month of your intended Spizellomyces transformation time.

[STEPS]
SECTION: Steps
1. On the bottom, divide an IM plate into quadrants.
SECTION: Steps
2. Draw circles 2.5 mm in diameter in each quadrant. 
 

SECTION: Steps
3. Place a 20-30 mm diameter glass culture tube into a 50 mL conical tube.
SECTION: Steps
4. Add enough 70% ethanol to cover the first inch of the culture tube.
SECTION: Steps
5. Burn off the ethanol on the tube using a flame, let the glass cool in the sterile area around the open flame.
SECTION: Steps
6. Dip the culture tube back into the ethanol.
SECTION: Steps
7. Repeat steps 3-5 a total of 3 times.
SECTION: Steps
8. Working within the sterile area around the flame, gently press the warm (but not enough to melt the agar), sterile culture tube into the agar at the center of one circle drawn earlier.
SECTION: Steps
9. Gently press on the agar and move the culture tube in circles until a depression is formed roughly the size of the circle drawn. 
 

SECTION: Steps
10. Repeat for all quadrants of all IM plates needed.
TITLE: Protocol for &#34;non-human primate necropsy&#34;
AUTHORS: jlanciego

[DESCRIPTION]
Here we describe the standard procedure conducted for performing a non-human primate necropsy, followed by removal of the brain and spinal cord from the skull and backbone, respectively.

[STEPS]
SECTION: Animal preparation
1. Pre-surgical anesthesia: to be induced with ketamine (5 mg/Kg) and midazolam (0.5 mg/Kg), administered intramuscularly
SECTION: Animal preparation
2. Terminal anesthesia: to be induced with an overdose of sodium pentobarbital (200 mg/Kg).
SECTION: Prefusion and pre-histological processing
3. Perform a T-shape incision with a scalpel through the thorax and abdomen.
SECTION: Prefusion and pre-histological processing
4. Open and retract the chest cavity to visualize lungs and heart.
SECTION: Prefusion and pre-histological processing
5. Open the left ventricle to allocate the infusion cannula at the level of the aortic
infundibulum and fixed it.
SECTION: Prefusion and pre-histological processing
6. Open the right atrium, getting started with the perfusion protocol at a constant
rate of 3,500 ml/h) with an infusion pump.
SECTION: Prefusion and pre-histological processing
7. First solution: 1,000 ml of saline Ringer solution.
SECTION: Prefusion and pre-histological processing
8. Second solution: 3,000 ml of a buffered solution made of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.125 M PB pH 7.4
SECTION: Prefusion and pre-histological processing
9.       
Third solution: 1,000 ml of a buffered solution made of 1% dimethylsulphoxide (DMSO) and 10% glycerin in 0.125 M PB pH 7.4
SECTION: Prefusion and pre-histological processing
10. Brain extraction: Once perfusion was completed, the brain and the spinal cord are removed from the skull and the backbone, respectively. 10 mm-thick brain blocks are made and the pia matter carefully removed under a dissection microscope. Brain blocks were stored for 48 h in a cryoprotectant solution containing 2% DMSO and 20% glycerin in 0.125 M PB 7.4
TITLE: Mycota Lab CTAB Protocol
AUTHORS: Stephen Douglas Russell

[DESCRIPTION]
This protocol is an amalgamation of the methods used by Osmundson et al. and Forin et al. for fungal herbarium specimens.

[STEPS]
SECTION: DNA Extraction Procedure
1. Create your stock Proteinase K and RNASE A solutions. Both at 20mg / mL. So for 100mg of Proteinase K, add 5 mL of molecular water. Same for the RNASE A. 
SECTION: DNA Extraction Procedure
2. Prep your CTAB buffer in 50mL tubes. Add 500 uL of CTAB buffer per sample. 96 samples add 48mL of CTAB.  For this amount of CTAB add 240 uL of Proteinase K and 240uL of RNASE A.
SECTION: DNA Extraction Procedure
3. Place fungal tissue and a stainless steel bead into a 1.5mL Eppendorf tube.
SECTION: DNA Extraction Procedure
4. Add 1mL of prewarmed (65◦C) CTAB extraction buffer with the addition of proteinase K (1 mg/mL) and RNase A (10 mg/mL).
SECTION: DNA Extraction Procedure
5. Apply three rounds of freeze-thaw consisting of alternating three min treatments in dry ice and a 70uC heating block, followed by a (30 minutes to overnight) incubation at 70◦C.
SECTION: DNA Extraction Procedure
6. Centrifuge for 10 minutes at max speed.
SECTION: DNA Extraction Procedure
7. Transfer the liquid phase to a new 1.5 mL microcentrifuge tube.
SECTION: DNA Extraction Procedure
8. Add 800 µL of phenol:chloroform:isoamyl alcohol (25:24:1). Invert the tube 15 times to mix.
SECTION: DNA Extraction Procedure
9. Centrifuge for 10 minutes at 10,000 rpm. 
SECTION: DNA Extraction Procedure
10. Move the upper liquid phase to a new 1.5mL tube and add 600 µL of chloroform:isoamyl alcohol (24:1). Invert the tube 15 times to mix.
SECTION: DNA Extraction Procedure
11. Centrifuge for 10 minutes at 10,000 rpm.
SECTION: DNA Extraction Procedure
12. Precipitate the DNA by adding ice-cold isopropanol (2/3 of the recovered volume) and incubate at −20◦C for 2 hours.
SECTION: DNA Extraction Procedure
13. Centrifuge for 10 minutes at 10,000 rpm. Pour off isopropanol.
SECTION: DNA Extraction Procedure
14. Add 300 µL of ice-cold 70% ethanol to wash. Let sit for 2 minutes.
SECTION: DNA Extraction Procedure
15. Centrifuge for 10 minutes at 10,000 rpm. Pour off the ethanol.
SECTION: DNA Extraction Procedure
16. Repeat the previous ethanol wash a second time.
SECTION: DNA Extraction Procedure
17. Let the pellet air dry for at least 15 minutes or until all the ethanol has evaporated.
SECTION: DNA Extraction Procedure
18. Resuspend the pellet in 20uL of molecular water. Incubate overnight?
SECTION: DNA Extraction Procedure
19. Bead cleanup. Add 20uL of beads to the tube. Wait 5 minutes. Put on magnet for 2 minutes. Remove liquid. Add 125mL of ethanol. Wait 2 minutes. Remove ethanol. Repeat wash. Let dry 1 minute.
SECTION: DNA Extraction Procedure
20. Add 20uL of molecular water.
SECTION: DNA Extraction Procedure
21. Store the DNA at -20C.
TITLE: bright field standard swarming imaging
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For imaging swarming behaviour of 40 young adult C. elegans on agar using the Phoenix multi-worm tracker system. Worms are synchronised by bleaching and refeeding for 72 hours, and then 40 young adult hermaphrodites are transferred by glass pipette onto a 35 mm regular NGM plate for imaging for 7 hours at 25 fps.</div></div>

[STEPS]
?. [Imaging plate preparation (Day -7)]
A separate batch of imaging plates is poured exactly seven days before each imaging day and stored at 4°C.
Imaging plates are 35 mm Petri dishes containing 3.5 mL low peptone (0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene) to limit bacteria growth.
?. [Imaging plate preparation (Day -1)]
Imaging plates are dried at 37°C overnight with the agar side down.
?. [Seeding imaging plate (Day 0, AM)]
The center of an imaging plate is seeded with a single 20 μL spot of cold diluted OP50 (OD=0.75) one to three hoursbefore imaging.
The overnight plate drying step allowed the bacteria to quickly dry atop the media in order to achieve a more uniform lawn by minimizing the “coffee ring” effect that would thicken the circular edge of the bacteria lawn.
?. [Bleach synchronising worms (Day -7 to -4)]
Bleach synchronise gravid hermaphrodites. Leave on rotator at 20 °C until use.
?. [Re-feed worms (Day -3, PM)]
Re-feed starved L1 worms onto 3-4 plates using a glass pipette. Incubate at 20 °C.
Culture plates are 55 mm Petri dishes containing 15 mL low peptone 0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene), and seeded with OP50.
?. [Imaging (Day 0)]
Forty animals are transferred by a glass pipette onto the imaging plate in a small drop of M9, away from the bacteria lawn.
?. [Imaging (Day 0)]
Imaging commences immediately following animal transfer in a liquid drop, on a custom-built six-camera rig (the Phoenix) equipped with Dalsa Genie cameras (G2-GM10-T2041). Seven-hour recordings with red illumination (630 nm LED illumination, CCS Inc.) are taken at 25 Hz using Gecko software (v2.0.3.1), whilst the rig maintain theimaging plates at 20 °C throughout the recording durations.
?. [Image data processing]
Images are segmented in real time by the Gecko software.
?. [Imaging (Day 0)]
Wash animals off of culture plates with M9 and collect in a 15 mL Falcon tube, wash in M9 twice, and aspire as much supernatent as possible after the last wash.
?. [Image data processing]
The recordings are manually truncated post-acquisition to retain aggregation and swarming dynamics only.
The start time is defined as the moment when the liquid dries and the all the worms crawl out from the initial location of the drop, and the end time is when the food is depleted and worms disperse with increased crawling speed.
TITLE: Soil sample(Citizen scientists, Chinese)
AUTHORS: Hsin-Mao   Wu

[DESCRIPTION]
Soil sample, citizen scientist

[STEPS]
SECTION: 採土
1. 選定地點，分為以下三種
醫院
公園
天然地區

SECTION: 採土
2. 移除表土，取距離土表5~7公分之處的土，裝入塑膠夾鏈袋中
SECTION: 採土
3. 將塑膠袋中的土均勻混合，倒入HDPE塑膠罐之中
 

SECTION: 採土
4. 將HDPE罐子收進袋子中
SECTION: 採土
5. 最後拍兩張照片
TITLE: GG1 - sgRNA cloning for Phaeodactylum tricornutum
AUTHORS: Mark Moosburner, Andrew Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The CRISPR-Cas9 gene mutagenesis system was adapted for the marine diatom Phaeodactylum tricornutum (CCAP-1055/1) (Figure 1).  Here, Cas9 and sgRNA(s) were delivered to Phaeodactylum by bacterial-conjugation transformation on an episome, or artificial chromosome, that is stably maintained and replicated independently from and with the Phaeodactylum chromosomal DNA.  Two sgRNA expression cassettes, sgRNA (1/1) and sgRNA (1/2) were cloned into the episome. The sgRNAs each contain a unique 20-nucleotide spacer sequence that , after sgRNA cassette transcription, guides the Cas9 to a nucleic acid target by complementary binding followed by Cas9 nuclease activity.  Two Phaeodactylum genes, Pt_GSII (Gene ID: 51092) and Pt_cGOGAT (Gene ID: 24739), were targeted by each sgRNA, respectively. The following protocol was followed to synthesize spacer sequences and sgRNA expression cassettes</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The presence and structure of EFNs in <em>Opuntia robusta</em> had not been investigated. We used light, scanning-electron, and transmission-electron microscopy to examine morphology, anatomy, and ultrastructure of the secretory spines in areoles in female and hermaphrodite individuals of <em>O. robusta</em>. Young cladodes develop areoles with modified and secretory spines as EFNs only active during the early growth phase of female and hermaphrodite individuals. EFNs are non-vascularized structures, with no stomata, that consist of three distinct tissues: a basal meristematic tissue; a middle elongation region; and an apical secretory cone (asc) formed by large globular epidermal cells, of sac shape, containing nectar and medullar elongated cells. TEM observations indicated the presence of Golgi apparatus, vesicles and plastids in the medullar and sup-epidermal cells of the asc, transporting nectar to the epidermal secretory cells. The nectar is transported through plasmodesmata, and then stored on the asc cells, to be secreted by breaking through the globular cells and pores.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Sampleholder preparation
AUTHORS: philippe.bechtold 

[STEPS]
?. The sampleholder contains 9 wells that are preloaded with an RT-qPCR Mastermix as follows:
?. Wells 1 to 3 contain probes and primers for the N1 target, wells 2 to 6 contain probes and primers for the N2 target, wells 7 to 9 contain probes and primers for the Human RP target. Wells 7,8,9 contain the respective positive control plasmid
?. The mastermix preloaded contains the following:  ,r  ,  ,  , filled up to   per well
[Geneon RT-PCR Mastermix (2x)]
[of each Primer]
[TaqMan Probe]
[BSA]
[MgSO4]
[with RNAse free water]
?. The sample holders with the mix are lyophilized with the following procedure: Freezing in   for  , transfer into precooled lyophilizer at   for  followed by transfer into a dissicator for heating up under vacuum.
-80 °C
-55 °C
[at least]
?. Sample holders are vacuum packaged for further use.
TITLE: noesypr1d_metab.nan
AUTHORS: NAN KB, John Glushka, Mario Uchimiya, Saraa Al Jawad, Christopher Esselman, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a protocol for running the Bruker pulse program "noesypr1d".

[BEFORE_START]
This protocol assumes your sample is loaded, locked, tuned, and shimmed in the magnet.

[GUIDELINES]
This protocol intends to provide concise instructions to carry out the experiment. For more comprehensive information, see Bruker's documentation including "Basic NMR Experiments" by clicking ? →  Manuals (docs) on the menu bar on TopSpin.

[STEPS]
SECTION: Create a new dataset
1. 
SECTION: Create a new dataset
1.1. On the menu bar on TopSpin, click on
Start → Create Dataset
 
 

SECTION: Create noesypr1d experiment file
0.29. When Lock, Tune, Shim are complete proceed to the specific protocol for your desired pulseprogram(s).
SECTION: Create a new dataset
1.4. A new window opens. On the right top bar, select
Source = /opt/NAN_METAB/par

In the list, select the one you want to use:
NOESYPR1D_br600_serum.par: Parameter set optimized for serum and plasma samples.
NOESYPR1D_br600_urine.par: Parameter set optimized for urine samples.

 


Click
OK
  
SECTION: Create a new dataset
1.2. Enter
NAME: Name of a set of datasets (e.g., human_serum_study1). Use a single string.
EXPNO: Dataset number. Use a positive integer.

Select
Directory: Your directory.

 
 

SECTION: Create a new dataset
1.3. Select
Read parameterset

Click the button
Select
29. 
29. 
28.1. pulsecal
27.2. rga
26.3. zg
28.1. 
SECTION: Acquire a spectrum
0.29. 
SECTION: Acquire a spectrum
2. Go to the "USE DEFAULT" tab below to proceed with the default optimized parameters.

3. 
3. 
3.2. Load the calibrated P1 using the following command in the command line.
 
(e.g., getprosol 1H 10.01 -7.45)

 

3.1. Select the AcquPars tab to show a parameter set.

 
 

3.3. Change parameters as needed.

Common parameters to adjust:
 

 

3.3. Click on
Acquire → Gain
in the menu bar to automatically set the receiver gain.

 
 

3.4. Click
Go
in the menu bar to acquire a spectrum.

 

3.1. Calibrate the 90° proton pulse (i.e., P1) using the following command in the command line.

 

A new window will appear to show the calibrated P1.
3.5. Click on
Process → Proc. Spectrum
in the menu bar to execute an automated processing macro.

 
3.2. Click the icon
_∏ _
to see a complete parameter set.

 

3.4. Acquire a spectrum. If more optimization is needed,   .
SECTION: Create a new dataset
1.5. Click
OK
3.6. If you want to modify parameters to improve your spectrum,   and move to a step case "MODIFY PAR".
TITLE: Pole Test
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to test mice in the Pole behavioural test.

[STEPS]
1. Habituate mice in the testing room for 60 minutes.
2. Place mice ~3cm from the top of the pole, facing upwards, and record their time to turn facing downwards and their time to reach the bottom of the pole. The mice have up to 1 minute to turn and 1 minute to reach the bottom before being returned to their home cage.
TITLE: WetLab-2 RT-qPCR procedure
AUTHORS: Jimmy Jung, Macarena Parra

[DESCRIPTION]
<p>Procedure used for RT-qPCR in the WetLab-2 study</p>

[STEPS]
?. [qPCR]
Preparation of qPCR reactions (per 25ul reaction):Promega GoTaq Probe Master Mix (2X): 12.5 ulForward Primer (20X): 1 ulReverse Primer (20X): 1 ulHydrolysis Probe (20X): 1 ulDNA Template: varied from 1 pg to 1ug per reactionMolecular Biology Grade Water: to 25 ul total volume
?. [RT-qPCR]
Preparation of RT-qPCR reactions (per 25ul reaction):Promega GoTaq Probe Master Mix (2X): 12.5 ulGoScript RT Mix for 1-step RT-qPCR: 5 ulForward Primer (20X): 1 ulReverse Primer (20X): 1 ulHydrolysis Probe (20X): 1 ulRNA Template: varied as listed (usually around 5ng)Molecular Biology Grade Water: to 25 ul total volume
TITLE: Squalene Quantification using Nile Red Staining (M4455 Version)
AUTHORS: Sebastian Triesch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This protocol is under development and for teaching purposes only!</span></div><div class = "text-block">Nile Red is a fluorescent dye that stains selectively hydrophobic substances. We assume that squalene accumulates in the cell membrane or in lipid vesicles where it can be stained by the dye. </div></div>

[STEPS]
?. Sample 1-2 ml Synechocystis culture, measure its OD at 750 nm and adjust it to 2 ml of OD (750 nm) = 0.2 in BG-11 media. Split your adjusted culture in 2x 1 ml. One portion will be stained with Nile Red, the other will serve as a negative control.
?. Stain one portion of the previously adjusted culture with c(final) = 10 µg/ml Nile Red.
Wear gloves and a lab coat when working with DMSO as it stains skin and cloth.
?. Incubate your culures for 30 min in the dark.
[Incubation]
?. Centrifuge all samples for 5 min at 5000 xg.
[Centrifugation]
?. Carefully discard the supernatant by pipetting. Avoid resuspending the pellet or losing cells! Try removing as much supernatant as possible.
?. Thoroughly resuspend all pellets in 1 ml BG-11 media.
?. Fill each 200 µl in 96 well plate's wells. Your sample volume should allow four wells per sample.
?. Measure each well's OD(750 nm) in the plate reader.
?. Measure Nile Red fluorescence. Use following wavelenghts: Excitation: 510 nm/Emission : 660 nm
?. Analyze your data: Normalize each well's fluorescence to the respective OD and subtract fluorescence without Nile Red from fluorescence with Nile Red. Compare your results to wild type Nile Red stained fluorescence.
TITLE: Seeding 90mm NGM plates with bacteria
AUTHORS: Cristian Riccio

[STEPS]
?. At the Bunsen burner, transfer the bacterial culture to a 50 ml Falcon tube in order to make it possible for the repeat combitip pipetter to reach the bacterial culture. Do so using a serological pipette and a pipetboy.
?. Set the repeat pipetter to dispense 600 ul at each push.
?. Aspire 10 ml of bacterial culture into a combitip.
?. Dispense 600 ul bacterial culture as a blob onto each 9 cm NGM plate.
?. Leave the plates to dry at room temperature over one or two days, with their lid closed and not near the Bunsen burner.
TITLE: Sampling macrofungi using fixed-sized plots
AUTHORS: Gregory M. Mueller, John Paul Schmit, Sabine M. Huhndorf, Leif Ryvarden, Thomas E. O'Dell, D. Jean Lodge, Patrick R. Leacock, Milagro Mata, Loengrin Umana, Qiuxin (Florence) Wu, and Daniel L. Czederpilz, Daniel L. Czederpiltz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol for sampling conspicuous macrofungi using fixed-size plots.</div></div>

[STEPS]
?. The investigator selects an area within the site for the permanent plot. The area should be representative of an important forest or grassland type at the site and should be chosen to optimize the diversity of habitat types sampled by the entire study (i.e., if the study covers grasslands, open woodlands, and dense forest, at least one plot should be set up in each habitat type). The plot should be established in as homogenous an area as possible and as easily accessible fro the road or trail as possible, without being susceptible to "edge effects" because it will be visited repitedly.
?. Each plot consists of 10 transects that are 100 m long. Typically, transects are laid out parallel to one another at 10-m intervals. If the shape of the area to be sampled does not allow that, however, some transects can be laid out end to end. Transects are marked every 5 m with a flag or stake. Each transect is assigned a unique letter, and each flag should be numbered sequentially within a transect (i.e. A1-A20; B1-B20; ...; J1-J20).
?. Each person sampling carries a plastic pipe or wooden pole and a rope that is 1.262 m long; the rope is used to circumascibe 5-m2 sictular subplots around each flag in a transect, giving rise to  20 5-m2 subplots per transect, for a total of 200 subplots, or a sampling area of 1000 m2 (0.1 ha) per plot. Care should be used to not walk in or unnecessarily disturb the subplots.
?. All macrofungi occurring in a subplot are collected, labeled with the transect letter and subplot number, and placed in an appropriate bag or container. At the end of the collecting day, specimens are transported back to the field station for sorting, describing, photographing, and drying. The substratum (soil, leaf litter, wood) is noted for each specimen.