eamag/protocols-with-steps huggingface dataset:
TITLE: DNA extraction protocol for the Bungarus multicinctus by using AxyPrepTM Multisource Genomic DNA Miniprep Kit
AUTHORS: Boyang Liu, Liangyu Cui, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
Genomic DNA was extracted from the muscle of a Bungarus multicinctus using the AxyPrepTM Multisource Genomic DNA Miniprep Kit (Axygen, China) with modifications as outlined in this protocol.

[STEPS]
1. The following protocol is a modification of the protocol by using the AxyPrepTM Multisource Genomic DNA Miniprep Kit. 

Approximately 10mg tissue was minced with sterilized scissors on ice.
on ice 

2. Add 350 µl Buffer PBS and 15 µl SDS Lysis Buffer and shake for 30s.
30 s 

3. Add 150 µl Buffer C-L and 20 µl Proteinase K. Immediately vortex for 1 min to mix well. After brief centrifugation, incubate at 56°C for 10 min.
1 min 
56 °C 10 min 
4. Add 350 µl Buffer P-D, vortex for 30s to mix well, and centrifuge at 12,000×g for 10 min.
30 s 
  

5. Place the DNA preparation tube in a 2 ml centrifuge tube, transfer the mixture in step 4 to the preparation tube, and centrifuge at 12,000×g for 1 min.
  

6. Discard the filtrate, put the preparation tube back into the original 2 ml centrifuge tube, add 500µl Buffer W1, and centrifuge at 12,000×g for 1 min.
  

7. Discard the filtrate, put the preparation tube back into the original 2 ml centrifuge tube, add 700µl Buffer W2, centrifuge at 12,000×g for 1 min.
  

7.1. In the same way, cleanse again with 700µl Buffer W2.
 

8. Discard the filtrate, put the preparation tube back into the original 2ml centrifuge tube, and centrifuge at 12,000×g for 1 min.
  

9. Place the DNA preparation tube in another clean 1.5 ml centrifuge tube, add 100-200µl Eluent or deionized water to the center of the preparation tube membrane, stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min to elute DNA.
1 min Room temperature
TITLE: Embedding and Cutting of Eye Tissue at UAB
AUTHORS: David       Anderson, Jeffrey  Messinger, Jamie Allen, Angela Kruse, Elizabeth Neumann, Nathan Heath Patterson, Richard       Caprioli, Kevin      Schey, Jeff Spraggins, Christine      Curcio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To describe the procedure for embedding and cutting eye tissue.</div></div>

[STEPS]
?. Prior to embedding, the posterior pole is trimmed to a 14-mm-wide belt of retina, choroid, and sclera containing major landmarks to help orientation.  This includes the optic nerve head, fovea, and horizontal meridian of the visuotopic map and extending anteriorly to pigmented tissue (ora serrata) at the edge of the ciliary body.
?. To stabilize the globe and standardize dissection, posterior poles are placed in an custom-designed, chilled aluminum 3” x 4” x 1” billet with a 30-mm-diameter hemispheric well and a set of parallel grooves 7 mm superior and inferior to the center of the well.
?. Globes are placed facing up in the well and the globe-billet combination oriented so the superior quadrant is positioned to the left and the grooves are vertical, thus making the long edge of the tissue block parallel to the optic nerve head and fovea axis. To guide a knife cut, the globe is snipped vertically (10 mm) with scissors (Roboz # 500216-G), guided by the parallel grooves. A tissue slicer (Thomas Sciences #6727C18) is placed in the two superior scissor cuts. With a guillotine motion, a superior cap is removed from the globe.While leaving the first blade in place to stabilize the globe, the inferior cap is removed by the same process at the second groove. The nasal periphery is removed with a slice 2 mm nasal to the optic nerve head (Pang et al., 2015).
?. Tissues are embedded in 2.6% carboxymethyl cellulose (Sigma C9481). Tissue belts are placed into cryomolds (Peel-A-Way 22 x 30 mm molds, Polysciences # 18646B) using a stereo microscope, oriented so the superior edge is facing up (sectioned first). Molds are filled with 5 ml of cold carboxymethyl cellulose and frozen at -20°C (NEED TO CHECK THIS FOR ERROR)(Anderson et al., 2020).
?. While cutting, histopathologic evaluation is used to assess location and quality, serial 10 µm cryosections were collected starting at the superior edge of the optic nerve head on pre-labeled 1x3 mm glass slides coated with 10% poly-L-lysine (Sigma Aldrich, St. Louis, MO, USA) and maintained at 37° during sectioning. Microscopy observations are made throughout the sectioning process.
?. Sections for MALDI IMS, MXiF, Codex, and proteomics are collected at predefined intervals depending on experimental design.
?. Samples are packaged for transport to Vanderbilt using a slide box vacuum-packed with an oxygen-absorbing packet within a Bitran freezer bag to help prevent oxidation and deterioration of lipid signalStored at -80°C before transport to Vanderbilt (Anderson et al., 2020)
TITLE: Long-read sequencing and data processing
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Long-read sequencing and data processing methods from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. Twelve TRAP samples and three TOTAL samples were sequenced using the Oxford Nanopore Technologies MinION platform. TRAP samples were equally divided by age and genotype (N = 3 per age:genotype). Library preparation was performed using the cDNA-PCR kit (SQK-PCS109). Raw fast5 data was basecalled and demultiplexed using Guppy (v4.5.2). Read data from FASTQ
files were aligned to the mm10 genome (Gencode M25 GRCm38.p6) using minimap2 (v2.18, RRID:SCR_018550)57. Transcript level quantification was then performed using Salmon (v1.4.0, RRID:SCR_017036)58.
TITLE: Post-Surgical Dissection of Uterine Body
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes dissection of the uterine body in preparation for 10X Visium, 10X Multiomics, pathology review, and biobanking.  Various systems can be used to sample and maintain the orientation of biospecimens collected from the uterus.  In this protocol we divide the uterus along its true axis and process the anterior and posterior walls of the uterus separately.  Other investigators have divided the uterus perpendicular to the true axis which captures both the anterior and posterior walls together at various points along the true axis.

[BEFORE_START]
Dilute phosphate buffered saline (Gibco; 14200-075) to 1X with nuclease-free water.

[STEPS]
1. Ensure you have a clean cutting surface and fresh, disposable surgical blades.
2. Remove uterus from ice and dry it.
3. Divide the cervix and uterus into halves in coronal or frontal place.
4. Identify internal cervical os.
5. Working on the anterior side of the uterine body, slice it into three horizontal sections from the internal cervical os as the lower limit to the fundus as the upper limit: fundus, anterior uterine body, and anterior lower uterine segment.

    

6. Remove the anterior lower uterine segment and place the remainder of the specimen on ice.
7. Divide the anterior lower uterine segment into five sections, from right to left.
8. Divide each section into four additional sections, from right to left.
9. Place each of the 20 anterior lower uterine sections on its side and using a razor blade, separate the serosa and myometrium from the submucosal myometrium and endometrium.
10. Using disposable weigh boats, weigh each of the 40 tissue specimen.
11. Tissue can be processed with protocols OCT-Embedded Tissue Preparation, Tissue Fixation Preparation, or Snap-Frozen Tissue Preparation depending on the desired downstream processing.
12. Repeat steps for anterior uterine body, anterior uterine fundus, posterior lower uterine segment, posterior uterine body, posterior uterine fundus.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This method is best used on colonies grown on solid media and subcultures in 7H9 liquid media. This method is high yeilding and is very efficient for short read NGS. It is not appropriate for direct tissue or fecal extraction. It is also not appropriate for liquid media containing high amounts of egg yolk. </p>
<p> </p>
<p>The use of trade, firm, or corporation names in this protocol is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture of any product or service to the exclusion of others that may be suitable.</p>

[STEPS]
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TITLE: LEGACY01: REGULATORY ISSUES
AUTHORS: Katrina M Pollock, Calliope Dendrou

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

[GUIDELINES]
ETHICS APPROVAL

The Study Coordination Centre will obtain approval from an NHS Research Ethics Committee (REC) and Health Research Authority (HRA). The study must also receive confirmation of capacity and capability from Imperial College Healthcare NHS Trust before accepting participants into the study or any research activity is carried out. The study will be conducted in accordance with the recommendations for physicians involved in research on human subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions.

CONSENT

Consent to enter the study must be sought from each participant only after a full explanation has been given, an information leaflet offered, and time allowed for consideration. Signed participant consent should be obtained. The right of the participant to refuse to participate without giving reasons must be respected. After the participant has entered the study, the clinician remains free to give alternative treatment to that specified in the protocol or to withdraw participant from lymph node sample donation at any stage if he/she feels it is in the participant’s best interest, but the reasons for doing so should be recorded. In these cases, the participants remain within the study for the purposes of follow-up and data analysis. All participants are free to withdraw at any time from the protocol treatment without giving reasons and without prejudicing further treatment.

CONFIDENTIALITY

Definition of terms 

Pseudonymised data can be linked to an individual e.g., through a coded study key kept at the NHS study site. 

Anonymised data cannot be linked directly back to an individual e.g., aggregated data for publication. 

The Chief Investigator will preserve the confidentiality of participants taking part in the study and is registered under the Data Protection Act.

Individuals will receive a single non-transferable study code when attending screening for the study and keep the same code upon enrolment to the study. Data collected for the study will be pseudonymised by means of a coded study key kept at the NHS study site (Imperial College Healthcare NHS Trust. Pseudonymised data will be transferred from the study site to Imperial College London and University of Oxford for analysis without the study key. Anonymised data will be published.

[STEPS]
TITLE: Creating Simulated Synapse Images
AUTHORS: Justin T Savage

[DESCRIPTION]
How to create simulated synapse images using FIJI.   

[STEPS]
SECTION: Prepare images
1. Copy some synapse images into a folder to serve as a source of synaptic puncta. Example images are available on Zenodo at DOI: 10.5281/zenodo.12191805
SECTION: Run ImageJ Macro
3. Open 20240312_sim_synapse_data_sparse.ijm from the SynBot GitHub by dragging it into FIJI
SECTION: Prepare images
2. Set up a larger folder for the code to use that contains a subfolder called "raw_images" that contains the images to use.
SECTION: Run ImageJ Macro
5. Choose the source folder prepared as explained in step 2.
SECTION: Run ImageJ Macro
6. Simulated images will be saved to the ouput folder created within the source folder from step 2. Text files are also written there which have the coordinates of the green and red puncta to serve as the ground truth for future analysis.
SECTION: Run ImageJ Macro
4. Change the noise multiplier in line 7 of the code if necessary. You can also adjust the minimum pixel size and threshold values to use in lines 13-20. These values should be stringent since you only want to use good puncta for making the simulated images and aren't trying to capture everything.
TITLE: SPARC Adenosine 2A Receptor Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment
AUTHORS: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the immunofluorescent labeling technique used to identify adenosine 2a receptor expression in CtB-labelled phrenic motor neurons and within a defined region of interest surrounding phrenic motor neurons.</div></div>

[STEPS]
?. Day 1: primary antibodies required:Adenosine 2A Receptor: Mouse anti-A2A (Millipore #05-717)Cholera toxin B-fragment: Goat anti-CT-B (Millipore #227040)
?. Place 40um transverse spinal cord sections into 1xPBS-Triton (0.1%) in 12 well cell culture plates
?. 3x washes in  1xPBS-Triton (0.1%) for 5 minutes each at room temperature
?. Antigen retrieval: place tissues into Heat-Induced Epitope Retrieval (TissuePro, Cat#: HIER01-32R) for 30 minutes at 85 degrees C
?. 3x washes in  1xPBS-Triton (0.1%) for 5 minutes each at room temperature
?. Blocking: place tissues in 5% Normal Donkey Serum (NDS) in 1xPBS-Triton (0.1%) for 60 minutes at room temperature
?. Primary Antibody Incubation: Incubate tissues in: 5%NDS in 1xPBS-Triton (0.1%), Mouse anti-A2A (1:1000), and Goat anti-Ct-B (1:2500) for 1 hour at room temperature
?. Continue primary antibody incubation overnight at 4 degrees C
?. Day 2: secondary antibodies required:AlexaFluor 488: donkey anti-goat (Invitrogen, Ref#A11055)AlexaFluor 594: donkey anti-mouse (Invitrogen, Ref#A21203)
?. 3x washes in  1xPBS-Triton (0.1%) for 5 minutes each at room temperature
?. Secondary Antibody Incubation: Incubate tissues in: 5%NDS in 1xPBS-Triton (0.1%), donkey anti-goat (1:1000), and donkey anti-mouse (1:500) for 2 hours at room temperature
?. 3x washes in  1xPBS for 5 minutes each at room temperature
?. Mount tissues on Superfrost Plus microscope slides
?. Allow slides to dry overnight
?. Coverslip with VectaShield Antifade Hard Set Mounting Medium (Cat#:H-1400)
TITLE: NGS grade DNA isolation from plant
AUTHORS: Anurag Daware

[DESCRIPTION]
NGS grade DNA isolation from plant (maize) leaf tissue 

[STEPS]
SECTION: Steps
1. Pre-heat water baths (65°C and 37°C) before beginning the extraction
SECTION: Steps
2. Prepare the extraction buffer as follows: 

5 mL 1 M Tris, pH 8 (final conc. 100 mM) 
2.5 mL 0.5 M EDTA (final conc. 25 mM) 
2.5 mL 5 M NaCl (final conc. 250 mM) 
0.5 g PVP-40 (final conc. 1% w/v)
40 mL Water
SECTION: Steps
3. Grind 100 mg of desiccated leaf tissue into a fine powder using bead beater
SECTION: Steps
4. Add 650 μl extraction buffer (EB) and 90 μl 10% SDS directly into each tube and incubate for 30 min at 65 0C while inverting the tubes 3-6 times.
SECTION: Steps
5. Add 250 μl KOAc to each tube and mix well by inverting several times. A white precipitate should form immediately.
SECTION: Steps
6. Spin for 5-15 minutes at 12,000 rpm at room temperature and then transfer 400-600 μl of the supernatant to a fresh tube.
SECTION: Steps
7. It’s recommended to spin again and move the supernatant to a fresh tube if for NGS purposes.
SECTION: Steps
8. Add an equal volume of isopropanol to the tube containing the supernatant. It’s recommended to prepare tubes filled with an equal amount of isopropanol in advance. At this point, samples can be left at -20 °C overnight.
SECTION: Steps
9. Spin samples for 5 minutes at 10,000 rpm and pour off the supernatant.
SECTION: Steps
10. Add 400-500 μl of 70-75% ethanol to each tube, then spin for 5 minutes at 7500 rpm at RT.
SECTION: Steps
11. Directly pour off the supernatant and air-dry samples completely. Tubes can either be left with caps opened on a rack at the bench or in a fume hood. It is recommended to put them upside-down on paper towels. This is an important measure to make sure the residual ethanol is gone as it can directly inhibit downstream enzyme activities. It is not recommended to remove liquid residuals with a pipette, as it may increase the chance of cross-contamination or loss of DNA.
SECTION: Steps
12. For genotyping, add 100-200 μl nuclease free water into each tube. There is no need to add RNase.
SECTION: Steps
13. For NGS purposes, it is important to add RNase, followed by one additional precipitation step (from No. 5 to 12), and to adjust the volume of water for each sample following quantitation to ensure uniformity.
TITLE: BTI mobile plant phenotyping system: PhenoRig and PhenoCage construction
AUTHORS: Li'ang  Yu, Magdalena M Julkowska

[DESCRIPTION]
Phenotyping Infrastructures are the basis to enable productive data collection and management.  Here - we describe how we constructed the PhenoRig and PhenoCage, the two cost-effective systems which can be constructed with lightweight materials to realize the high-throughput image collection to phenotype plant growth in a growth chamber or a greenhouse workspace.    

[STEPS]
SECTION: Overview
1. This protocol is a part of BTI mobile plant phenotyping system (https://github.com/Leon-Yu0320/BTI-Plant-phenotyping). In this part, we'll introduce constructing a set of lightweight phenotyping facilities paired with the phenotying computational pipelines we developed. 
SECTION: Materials
2. Construction of phenoRig and phenoCage
To increase the mobility of phenotyping facilities, lightweight materials such as wood bars and aluminum bars will be recommended. Also, facilities can be disassembled into smaller pieces for storage when anchoring pieces with hexagon socket screws or other types of screws. See two examples below for details: 

 
  

 

SECTION: Installation of Raspberry Pi computers
3. 
 Raspberry Pi computers will be anchored to both two facilities. We built the device based on the relative size of Arabidopsis plants and cowpea plants and the sizes of each component used for construction were listed in the following tables. Installations of Raspberry Pi cameras and computers are shown as photos below. Details about construction materials, size, and reference cost can be reached in Table 1 and Table 2 . 
   



SECTION: Installation of Raspberry Pi computers
4.
TITLE: UF H&E Staining
AUTHORS: Marda Jorgensen, Franchesca Farris

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Hematoxylin and Eosin stains are the prefered method for histopathologic assessment of tissue sections.</div><div class = "text-block">Hematoxylin is used to illustrate nuclear detail in cells. The resulting intensity and shade of the dye in the cells is primarily reliant on the length of time the sample spends in hematoxylin. The cytoplasmic component of the section is identified by Eosin, which is the most commonly used counterstain in histology. It's varying shades of pink and red provide an exceptional contrast between the nuclear and cytoplasmic areas of interest. </div></div>

[STEPS]
?. Prepare staining line with reagents in order of use, based on steps three through 25.
?. Place slides into a xylene compatible slide rack.
?. Immerse slide holder in the first xylene staining station for 5 minutes.
?. Immerse slide holder in the second xylene staining station for 5 minutes.
?. Remove slide holder from xylene and transfer to the first staining station of 100% EtOH for 5 minutes.
QUickly blot excess xylene from rack before transfering into ethanol.
?. Transfer slide rack into the second EtOH station for an additional five minutes.
?. Transfer to 95% EtOH for 3 minutes.
?. Transfer to 70% EtOH for 3 minutes.
?. Transfer to 50% EtOH for 3 minutes.
?. Transfer to water for 2 minutes.
?. Transfer to the second change of water for an additional 2 minutes.
Quickly blot excess water from slide holder before going into hematoxylin.
?. Transfer slides to Hematoxylin for 1 minute and 15 seconds.
?. Rinse in de-ionized water,  and then transfer slides to tap water for 30 seconds.
?. After rinsing in tap water, place slide rack back into de-ionized water for 1 minute.
?. Transfer to second water staining dish for 1 minute.
?. Transfer to Aqueous Clarifier for 45 seconds.
?. Do a quick rinse in de-ionized water and then transfer to tap water for 1 minute.
?. Transfer to bluing reagent for 1 minute.
?. Place in water for 5 minutes.
?. Transfer to 80% EtOH for 1 minute.
?. Transfer to Eosin-Y for 1 minute.
?. Transfer to 100% EtOH for 2 minutes.
?. Transfer to the second 100% EtOH station for 5 minutes.
Be sure to blot excess ethanol before going into xylene.
?. Transfer to xylene for 5 minutes.
?. Transfer to a fresh xylene station for 5 minutes.
?. After the second xylene change, use a xylene compatible mountant to affix a coverslip to the slide.
TITLE: Transmission electron microscopy protocol for anaerobic ciliates
AUTHORS: Johana Rotterova, Ivan Čepička

[STEPS]
?. [Fixation]
Isolate 1 ml of thriving culture of ciliate cells, fix with 2.5% (v/v) glutaraldehyde (Polysciences) and subsequently centrifuge at 800×g for 5 min at 4 °C.
?. [Fixation]
Carefully replace supernatant with 1 ml of 2.5% (v/v) glutaraldehyde in 0.2M SCB (Sodium Cacodylate buffer, pH 7.2) and incubate 1 hour on ice.
?. [Fixation]
Then, rinse the pellet carefully with SCB three times per 15 minutes.
?. [Fixation]
Postfix the cells with 1% OsO4 in distilled water for 1 hour on ice.
?. [Rinsing and embedding]
Rinse the cells with distilled water (5 minutes), dehydrate them in a graded ethanol series from 30% to absolute ethanol: through 30%, 50%, 70%, 80%, 90%, 95%, and finally three times in absolute ethanol, each per eight minutes.
?. [Rinsing and embedding]
Let the embedded cells get polymerized at 70 °C for 48 hours.
?. [Cutting ultrathin sections]
Cut serial ultrathin sections on an Ultracut E ultramicrotome (Reichert) using a diamond knife and stain with lead citrate and uranyl acetate (2–3%).Protocol is adapted from Rotterova et al. (2018) and Beinart et al. (2018).
?. [Rinsing and embedding]
Incubate cells two times in 1:1 ethanol-acetone mixture per 15 minutes.
?. [Rinsing and embedding]
Incubate cells once in 2 ml of 100% acetone per 15 minutes, and finally substitute aceton with acetone-resin mixture (EPON resin Poly/Bed 812, Polysciences) and incubate per 6-12 hours.
?. [Rinsing and embedding]
Substitute absolute EPON resin (Poly/Bed 812, Polysciences) three times and finally embed cells in in the resin into capsules.
TITLE: Cleaning up a biohazardous spill in a biosafety cabinet
AUTHORS: Steven Wilhelm, Gary LeCleir, Ashley Humphrey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">BIOLOGICAL SPILL RESPONSE </span><span>method to be used in the event of a biosafety incident. </span></div></div>

[STEPS]
?. Let the BSC run. Do not turn off.
?. Remove broken glass with forceps, tweezers or other tools and place glass in a sharps container.  Do not wipe up broken glass.
?. Cover spill with paper towels.
?. Pour (don’t spray) disinfectant to contaminated surface by pouring it around the periphery of the covered spill moving inward.  Allow the appropriate contact time for the disinfectant and agent.
?. After the contact time, wipe up the spilled material.
?. Reapply disinfectant to the affected area and after the appropriate contact time, wipe up the area. Repeat if necessary.
?. Perform disinfection before removing items.
?. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.