eamag/protocols-with-steps huggingface dataset:
TITLE: Colony PCR
AUTHORS: Georgina Elisa Diego Macías, Ana Belem García González, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
Protocol for a PCR from a colony

[STEPS]
1. Dissolve one colony in 20 µL of nuclease-free water and heat at 80°C for 10 minutes.

2. Prepare the following 50 uL reaction in a 0.2mL PCR tube on ice:
 

3. Gently mix the reaction and spin down in the microcentrifuge
4. Cycling conditions for a routine PCR:
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. Click on
Acquire → Prosol → Single scan pulse calibration (pulsecal) 
to calibrate the 90° proton pulse (i.e., P1).
 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: Unlocking Spatial Molecular &amp; Cellular Relationships of SARS-CoV-2 in Archived Human Tissue
AUTHORS: Sam Pattle, Jenna Gregory, Fergal M Waldron

[DESCRIPTION]
The true prevalence of SARS-CoV-2 infection in the UK population remains unknown. Phylogenetic diversity estimates derived from genomic sequence tracking algorithms suggest possible arrival of viral strains in the UK several weeks earlier than the first confirmed cases. Diagnostic pathological specimens have continued to be recovered from patients throughout the Covid-19 pandemic, as part of standard investigation and workup across the normal range of clinical indications. Given the symptomatic heterogeneity and probable high rate of asymptomatic infections, we hypothesise that tissue biopsies have already been taken from living patients with clinically unrecognised SARS-CoV-2-infections. Here, we describe the protocol for a retrospective cohort study with the aims of i) detecting SARS-CoV-2 using qRT-PCR in archived biopsy tissue recovered from patients over the course of the pandemic to date, ii) demonstrating qualitative and quantitative evidence of SARS-CoV-2 tissue tropism and cellular-molecular patterns of infection using histological, in situ hybridisation and immunohistochemical techniques, and iii) linking those spatial molecular-cellular characteristics of SARS-CoV-2 infection to clinico-pathologic parameters. The results will provide new insights into the tissue tropism and rates of asymptomatic and ‘atypical’ SARS-CoV-2 infections - information that could immediately impact healthcare practices or public health policy.

[STEPS]
TITLE: Microbiome Assay
AUTHORS: Priota Islam

[STEPS]
?. Preparing worms Chunk N2s on 3-4 plates on Wednesday, also book the hood for next Wednesday morning to dry the seeded plates Bleach the N2s on Friday and keep the tube in the rotator inside the 20C incubator On Monday refeed the arrested L1s to be young adults on Thursday (Tracking Day1), do the same on Tuesday for the worms to be young adults on Friday (Tracking Day2)
?. Preparing Bacterial culture On Tuesday, grow an overnight culture of the bacteria to be tested by inoculating a single colony in LB broth (1 single colony+100ml LB Broth), in the shaking incubator at 200-220rpm On Wednesday morning, take the culture off the incubator and seed 2 maintenance plates per strain with 100ul of the liquid culture, leave the plates under the hood to dry Store the rest of the bacterial culture in a falcon tube at 4C to be used the following day for tracking i.e. Thursday Repeat steps 1-3 on Wednesday for tracking on Friday.
?. Pre-conditioning L4s The day before tracking Wednesday/Thursday, wash 2 plates of worms refed either Monday/Tuesday respectively with M9 and spin at 2500 for 2 mins Remove the supernatant with a Pasteur pipette leaving about 250ul in the tube Pipette about 20ul of the L4s on to the seeded plates with the bacteria to be tested and leave at 20C overnight
?. Tracking 1. On the day of tracking measure the OD of the bacterial culture stored at 4C 
2. Match the starting ODs for the cultures being tested on the same day and note them down 
3. Measure the weight of three imaging plates and record the average 
4. Label the imaging plates according to the tracking plan 
5. Seed 12 plates with 20ul of each bacterial culture (6 plates for the preconditioned L4s and 6 plates for young adults) and leave to dry 6. For the young adults: 
  -Wash 2 plates with M9 and spin at 2500rpm for 2mins 
  - Remove as much supernatant as possible and pipette the remaining worms on to an unseeded maintenance plate 
  -Once the M9 has dried, pick 5 adult worms onto each imaging plates and image right away 7. For the preconditioned L4s, just pick from their individual plates and image right away
TITLE: TMT labelling
AUTHORS: Louise Uoselis

[DESCRIPTION]
TMT labelling

[STEPS]
1. Reconstitute lyophilised peptides in 25 µL by vortexing each sample for ~10 s, and then leaving to sit at Room temperature for 15 min. Each sample was then sonicated for 5 min in a waterbath sonicator in an ice slurry. 

2. Determine the peptide concentration of each sample spectroscopically
3. Aliquot10 µg of peptides for each sample into a LoBind microfuge tube and add 100 mM triethylammonium bicarbonate to a final volume of 100 uL for each sample. 

4. For the pooled batch control, divide 100 µg by the total number of samples, and aliquot that amount of peptides from each sample into one tube. Make up the pooled control volume to 100 uL with 100 mM triethylammonium bicarbonate (can lyophilise the samples to reduce the volume if the total pooled volume exceeds 100 uL). 

5. Bring the TMT labels to Room temperature. 

6. Reconstitute each label in acetonitrile as per the manufacturer’s instructions. 
7. Add the required volume of the designated label to each sample, and vortex each sample for ~ 5 s to mix. 

8. Leave samples to incubate at Room temperaturefor60 min(static)

9. Add hydroxylamine to a final concentration of 26%/sample, vortex each sample for ~5 s, and incubate at Room temperaturefor15 minto quench the TMT labelling reaction

10. Combine the samples into their designated batches, and lyophilise the pooled samples. Seal each tube with parafilm and store at  -80 °C  for downstream processing.
TITLE: Immunostaining of Bodo saltans
AUTHORS: Ewa  Chrostek,   Mastaneh Ahrar,   Gregory Dd Hurst

[DESCRIPTION]
This protocol is used in our Laboratory in Liverpool to perform IF on Bodo saltans cells. 

[STEPS]
SECTION: Culture conditions
1. Bodo saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na2HPO4)1. Cultures were incubated at 22 °C in T25 tissue culture flasks containing 20 ml of media bacterized with Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700831). 
SECTION: Immunostaining
2. Please, follow the steps below to prepare cells for immunostaining.
SECTION: Immunostaining
2.1. Filter the culture through 100 and 8 µm filter.
SECTION: Immunostaining
2.2. Harvest the cells by centrifugation at 1200 × g for 12 mins at 19 °C.
SECTION: Immunostaining
2.3. Wash the cells with PBS and centrifuge as described above. 
SECTION: Immunostaining
2.4. Dissolve the pellet in 15 µl of PBS and mix with the same volume of low melting temperature agarose (eg. Thermo Fisher Scientific) in a single well of a 96-well plate. Let it ser for a few seconds.
SECTION: Immunostaining
2.5. Add 200 µl of 4% PFA and incubate at room temperature for 10 minutes or at 4 °C overnight.
SECTION: Immunostaining
2.6. Wash with PTX (PBS + 0,1% TritonX) 4 times, 30 mins each wash.
SECTION: Immunostaining
2.7. Block in 5% FBS+PTX overnight at 4 °C.
SECTION: Immunostaining
2.8. Incubate with primary antibody (1:100, diluted in PTX + 5% FBS), for 5 hours at room temperature or overnight at 4 °C. 
SECTION: Immunostaining
2.9. Rinse and wash 3 times for 1 hour with PTX at room temperature. 
SECTION: Immunostaining
2.10. Incubate with secondary antibody (1:1000, diluted in PTX + 5% FBS) overnight at 4°C.
SECTION: Immunostaining
2.11. Rinse and wash 3 times for 1 hour with PTX at room temperature. During 2nd wash add Hoechest 33342 (Thermo Fisher, 1:2000) for 10 minutes. This will be rinsed away with the 3rd wash.
SECTION: Immunostaining
2.12. Remove the agarose from the well with clean forceps and place it on a microscope slide. 
SECTION: Immunostaining
2.13. Add a drop of a mounting medium (eg. Vectashield, Vector Laboratories), and flatten the agarose as much as you can using the coverslip. 
SECTION: Immunostaining
2.14. Proceed with either fluorescence or confocal imaging.
SECTION: Immunostaining
2.15.   

SECTION: Immunostaining
2.16.
TITLE: HYPROP soil sample collection
AUTHORS: marshall.bennett

[DESCRIPTION]
Procedure developed using information gathered from the following sources: 

Shokrana, M. S. B., & Ghane, E. (2020, February 22). Measurement of soil water characteristic curve using hyprop2. Measurement of soil water characteristic curve using HYPROP2. Retrieved February 21, 2023, from https://www.sciencedirect.com/science/article/pii/S2215016120300601 

METER Group. (n.d.). HYPROP 2. Operation Manual - HYPROP 2. Retrieved February 21, 2023, from http://library.metergroup.com/Manuals/18263_HYPROP_Manual_Web.pdf 


[STEPS]
1. Gather tools needed for HYPROP sample ring collection. 
2. Identify sampling locations. Collect and record all appropriate field data (GPS coordinates, soil texture information, weather, etc.). 
3. Clear the surface of the soil to remove excess plant cover or debris. DO NOT DISTURB THE SOIL It is important to note that soil samples must remain undisturbed during collection.   

4. Remove both caps from the HYPROP ring.  

5. Place the sample ring with the cutting edge facing down firmly onto the flat surface of the soil.   

8. Remove excess soil using a blade or the trowel, clean the outer surface of the HYPROP sample ring with a rag or paper towels, and place both caps on the ends of the ring.     

6. Use a mallet and the HYPROP sample ring insertion tool or a piece of wood to drive the HYPROP sample ring completely into the ground. DO NOT COMPRESS THE SOIL INSIDE OF THE RING       

7. Use a trowel to dig around the outer edge of the HYPROP sample ring. Do not disturb the soil inside of the ring. Once the bottom of the ring has been exposed, without the soil inside of the ring being disturbed, use a flat blade or trowel to scoop the sample from underneath.
TITLE: Sectioning of Mouse Brain by Microtome
AUTHORS: Maryana Nissan

[DESCRIPTION]
This protocol describes how to use the microtome to prepare and slice mouse brain sections for Immunohistochemistry 

[STEPS]
SECTION: Preparation Methods
1. While preparing the microtome, make sure the blade is covered. The microtome used in the lab is from Leica Microsystems. 
SECTION: Preparation Methods
2. Slide the plate into the stage and secure it by tightening it with a screw. 
SECTION: Preparation Methods
3. Orient the plate so that it is centered.
 
SECTION: Preparation Methods
4. Add a few drops of 90% of ethanol on the sides of the plate and place dry ice to cool the microtome. The microtome will be cooled and ready for usage when the plate accumulates ice. 
SECTION: Preparation Methods
5. When the microtome is cooled, add a big drop of 1X Phosphate-Buffered solution [1X PBS] to the center of the plate 
SECTION: Preparation Methods
6. Quickly orient the brain on top of the 1X PBS bubble, ensuring that it is centered. 
SECTION: Preparation Methods
7. Double check the brain is in the center and straight. When it is, add more 1X PBS to secure it. 
SECTION: Preparation Methods
8. Add powdered dry ice over the brain to cool it. Let the brain cool for 2-5 minutes prior to slicing. 
SECTION: Preparation Methods
9. Prior to slicing, adjust the thickness setting. This knob is found on the side of the microtome. 
SECTION: Preparation Methods
10. Prepare the 24-well culture plate (Thermo Scientific, CAT # 142475] with cryoprotectant fluid. Brain sections will go here after they are sliced. 
SECTION: Slicing Methods
11. Remove the protective cover of the blade. 
SECTION: Slicing Methods
12. Move the stage up, placing the brain just below the blade. 
SECTION: Slicing Methods
13. Slowly move the blade towards you, slicing a section of the brain. To prevent the sections from folding and breaking, slice slowly and in a single continuous motion. 
SECTION: Slicing Methods
14. Gently scoop the brain section away from the blade, using a thin painting brush. 
SECTION: Slicing Methods
15. Slowly place the brush into cryoprotectant fluid. Gently move the brush to allow the section to unfold in the liquid. 
SECTION: Slicing Methods
16. Press the button on the side of the microtome to push the stage 1 point higher. This will allow you to slice the next brain section, at the thickness you have set. 
SECTION: Slicing Methods
17. Continue slicing until you have sliced the entirety of the brain. 
SECTION: Slicing Methods
18. Secure the 24-well plate with tape. Store brain sections in cryoprotected fluid at -20C.
TITLE: 4% PFA + 2.5% Glutaraldehyde Fixative
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare 4% Paraformaldehyde (PFA) + 2.5% Glutaraldehyde Fixative. This is utilized to fix the tissue after electrophysiology recordings. 

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Immunohistochemistry on mouse brain sections
AUTHORS: Shiyi Wang

[DESCRIPTION]
Immunohistochemistry on mouse brain sections

[STEPS]
1. **Anesthesia and Perfusion**
1.1. - Anesthetize mice with 200 mg/kg Avertin.
1.2. - Perform transcardial perfusion using TBS/Heparin followed by 4% paraformaldehyde (PFA).
2. **Brain Collection and Post-Fixation**
2.1. - Remove the brain and post-fix it in 4% PFA overnight at 4°C.
3. **Cryoprotection and Storage**
3.1. - Cryoprotect the brain in 30% sucrose solution until sunk.
3.2. - Embed the brain in a solution containing 2 parts 30% sucrose and 1 part O.C.T. (TissueTek).
3.3. - Store the brain blocks at −80°C until sectioning.
4. **Sectioning**
4.1. - Section the brain into 30 μm, 40 μm, or 100 μm thick coronal slices using a cryostat.
4.2. - Store the sections in a 1:1 mixture of TBS and glycerol at −20°C for further use.
5. **Preparation of Sections**
5.1. - Wash the brain sections in 1x TBS containing 0.2% Triton X-100 (TBST).
6. **Blocking**
6.1. - Block non-specific binding by incubating sections in 10% normal goat serum (NGS) diluted in TBST for 1 hour at room temperature.
7. **Primary Antibody Incubation**
7.1. - Incubate sections with primary antibodies diluted in blocking buffer (10% NGS in TBST) for 2-3 nights at 4°C with gentle shaking.
7.2. - Primary antibodies used: - Anti-LRRK2 (Rabbit, 1:500; ab133474, Abcam), HA (Rat, 1:500; 11867423001, Roche), Phospho-ERM (Rabbit, 1:500; 3141, Cell Signaling), Sox9 (Rabbit, 1:500; AB5535, Millipore), GFAP (Rabbit, 1:500; Z0334, Agilent DAKO), VGluT1 (Guinea pig, 1:2000; 135304, Synaptic Systems), PSD95 (Rabbit, 1:300; 51-6900, Innovative Research), VGAT (Guinea pig, 1:1000; 131004, Synaptic Systems), Gephyrin (Rabbit, 1:1000; 147011, Synaptic Systems)
8. **Secondary Antibody Incubation**
8.1. - Wash sections with TBST.
8.2. - Incubate sections in Alexa Fluor conjugated secondary antibodies diluted 1:200 in TBST for 2-3 hours at room temperature.
9. **Mounting**
9.1. - Wash sections in TBST.
9.2. - Mount sections onto glass slides using a homemade mounting media (90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate).
9.3. - Seal the edges of the coverslip with nail polish.
10. **DAPI Staining**
10.1. - Add DAPI (1:50,000) to the secondary antibody solution for the final 10 minutes of incubation.
11. **Imaging**
11.1. - Acquire images using a fluorescence microscope (e.g., Olympus FV 3000).
TITLE: 3.7% Paraformaldehyde solution
AUTHORS: Institute of Medical Biotechnology

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for preparing a 3.7% Paraformaldehyde (PFA) solution. 3.7% PFA solution can be used to fix tissues. </div></div>

[STEPS]
?. For , add  to a glass beaker on a stir plate.
[3.7% Formaldehyde]
[1X DPBS]
Be sure to work under a ventilated fume hood.
?. Heat while stirring to approximately .
55 °C
?. Add  to the heated DPBS solution.
[paraformaldehyde powder]
?. Keep stirring until all PFA powder is dissolved.
Take care that the solution does not boil to prevent formic acid formation.
For better dissolubility of PFA, you can add NaOH -- one drop at the time.
?. Once all PFA powder is dissolved, adjust the pH to  using HCl.
?. Add DPBS subsequently until the total volume of  is reached.
1 L
?. Transfer the solution into tubes of the desired proportions and store at  until use.
-20 °C
TITLE: Genome-wide quantification of TF binding at single DNA molecule resolution
AUTHORS: Rozemarijn    Kleinendorst, guido.barzaghi , Mike    L. Smith, Judith    B. Zaugg, Arnaud   Krebs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements (CREs). We recently developed Single Molecule Footprinting (SMF) to simultaneously resolve the occupancy of multiple proteins including Transcription Factors (TFs), RNA Pol II (Pol II) and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve TF binding patterns at CREs. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 days. Analysis can be performed in 2 days using a dedicated R package. The protocol can be executed in any laboratory with access to high- throughput sequencing. Our method can be used to analyse how TFs cooperate and antagonize to regulate transcription. 


</div></div>

[STEPS]
TITLE: Protocol for assembling SARS-Cov-2 runs  with s-aligner
AUTHORS: Juanjo Bermúdez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for using Contignant s-aligner to de-novo assemble SARS-Cov-2 genomes. S-aligner outperforms common open-source software for de novo-assembly of viruses by 110% increased performance.</div></div>

[STEPS]
?. [Indexing and fast-testing]
?. [Indexing and fast-testing]
results/your-run-id-500.fa should have the largest contig bigger than 1.200bp and at least one of the files containing aligned reads to form a contig should look like that:
?. [Indexing and fast-testing]
?. Trim your data and start again.
?. [Indexing and fast-testing]
?. [Indexing and fast-testing]
and continue as if the contig length is >1.200bp
?. [Indexing and fast-testing]
Discard this dataset or look for some tool that can filter your data.
?. [Indexing and fast-testing]
but index a larger fraction of the dataset.
?. [Indexing and fast-testing]
increasing the number of reads until you get a satisfactory assembly.
?. [Indexing and fast-testing]
Use QUAST to get statistics.
?. [Indexing and fast-testing]
Try scaffolding the contigsThen use QUAST with the result
?. [Indexing and fast-testing]
?. [Indexing and fast-testing]
Congratulations. You are done.
?. [Indexing and fast-testing]
Congratulations. You are done.
TITLE: RNA Isolation from Plant Tissue Protocol 1: Qiagen RNeasy Plant Mini Kit
AUTHORS: Jim Leebens-Mack, Charlotte Carrigan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol implemented by: Jim Leebens-Mack and Charlotte Carrigan </div><div class = "text-block"><span>The first protocol used for isolation of total RNA was Qiagen’s </span><a href="https://www.protocols.io/view/protocol-1-qiagen-rneasy-plant-mini-kit-4vxgw7n/materials" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">RNeasy Plant Mini Kit</span></a><span> (Qiagen, Valencia, CA). These kits are commercially available and widely used in RNA isolation. For detailed methods associated with this kit we refer readers to the “</span><a href="https://www.qiagen.com/es/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">RNeasy Mini Handbook</span></a><span>” available for download at </span><a href="http://www.qiagen.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">www.qiagen.com</span></a><span>. </span></div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div></div>

[STEPS]