eamag/protocols-with-steps huggingface dataset:
TITLE: Hybrid protocol for Nuclei Isolation and 10X Genomics Single Cell 5&#39; Gene Expression for Human Ovary Explants
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics Single Cell 5' Gene Expression hybrid protocol using nuclei suspension isolated from fresh, frozen human ovary explants. The Chromium X  (PN-1000331) was used for Gem Generation, and the Chromium Connect (PN-1000171) was used for preparing Gene expression libraries from cDNA inputs. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Nuclei Isolation Protocol for Human Ovary Explants
1. Chapter 1—Single Cell Gene Expression & Chromium Fixed RNA Profiling of the protocol CG000505 REV A was used to isolate nuclei from frozen human ovary explants with the following modifications: 1) a cordless motor pestle (VWR, Catalog number 47747-370) was used for Step f, Page 30 and 2) the samples were incubated for 15 min on ice for Step h, Page 30. 

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/sample-prep/chromium-nuclei-isolation-kit-sample-prep-user-guide


SECTION: Chromium Next GEM Single Cell 5&#x27;Reagent Kits v2 (Dual Index). GEM Generation, Barcoding, Post GEM-RT Cleanup and cDNA Amplification.
2. Step 1 and step 2 of protocol CG000331 REV E were used for GEM Generation, Barcoding, Post GEM-RT Cleanup, and cDNA Amplification. The Chromium X was used for Gem Generation. 

https://www.10xgenomics.com/support/single-cell-immune-profiling/documentation/steps/library-prep/chromium-single-cell-5-reagent-kits-user-guide-v-2-chemistry-dual-index




SECTION: Chromium Next GEMSingle Cell 5&#x27;Reagent Kits v2 (Dual Index). Gene expression library construction.
3. The protocol  CG000474 Rev B was used for Automated Gene expression dual index library construction from cDNA inputs. 

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/automated-gene-expression-library-construction
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Fluorescent labeling of reovirus virions with succinimidyl ester-conjugated fluorescent probes</p>

[BEFORE_START]
<p>You will need purified reovirus and dimethylformamide.</p>
<p>Sodium bicarbonate must be made on the day of labeling.</p>
<p>SE-conjugated dyes are not limited to Alexa dyes.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Splitting p1 (1xT75) to p2 (2xT150)
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol includes splitting growing cell line  from a T75 flask  into two T150 flasks for expansion. </div></div>

[STEPS]
?. [Observations]
At 90-100%  confluency, split T75 flask into 2xT150 flasks
?. [Preparation]
Turn off UV lights and clean hood with 70% ethanol
?. [Preparation]
Clean items with 70% ethanol and bring into hood    a. DPBS -/-     b. Sterile Filtered Media     c. Label 2xT150 flasks with ID, date, and p2, add 28 mls   of media per flask    d. 0.05% Trypsin
28 mL
?. [Culturing]
Aspirate old media
?. [Culturing]
Rinse by adding 5 mls   of DPBS  per flask and gently swirling
5 mL
?. [Culturing]
Aspirate DPBS
?. [Culturing]
Add 2 mls   of trypsin per flask and place in incubator   for 2-3 mins   until cells are stating to detach     a. Can also gently tap the side of the flask to detach cells
2 mL
37 °C
?. [Culturing]
Check under microscope
?. [Culturing]
Add 10 mls  of fibroblast media per flask to inactivate trypsin
10 mL
?. [Culturing]
Collect cell suspension in a 15 ml conical
?. [Culturing]
Spin at 600 rpm    for 4 mins
Centrifuge: 600 33
?. [Culturing]
Aspirate supernatant, being careful to not aspirate the pellet
?. [Culturing]
Tap the pellet to resuspend
?. [Culturing]
Add fibroblast media to 4 mls
4 mL
?. [Culturing]
Add 2 ml  of cell suspension per flask
2 mL
?. [Culturing]
Place flask in incubator (5% CO2 , 37oC  )
37 °C
?. [Culturing]
Observe and feed the next day, then feed every 2-3 days until 90-100% confluent
?. [Clean Up]
Throw away biohazard materials properly
?. [Clean Up]
Clean and sterilize hood with 70% ethanol and turn on UV
?. [Clean Up]
Update cell culture notes in lab notebook
TITLE: Intravenous injections
AUTHORS: Goran  Tomic

[DESCRIPTION]
Protocol for mouse tail-vein injections of cell lines. 

[BEFORE_START]
I block randomise* the injections when multiple groups/treatments are present (e.g. Ctrl and KO or isotype control/depleting antibody). The goal is for each cage to have all of the cell lines/treatments.
 Starting with Cage 1, I inject different cell lines assigned to each mouse within that cage, and then move to the next cage. This way, any bias with cells sitting on ice for extended periods of time is applied equally across all the cell lines used.

 *Block randomisation prevents the situation where all mice within one cage receive the same cell line/treatment. The way I do it: A random number is assigned to each mouse in a cage (block), and the treatments assigned from lowest to highest number within each cage (e.g Ctrl1 to lowest number, Ctrl2 for the second lowest, etc.). Random number is generated using the ‘rand’ function in Excel, but copied and pasted as ‘values’ so they would not change every time.

[STEPS]
1. Grow the cells 48-72 h before injection (around 70-80% confluent).
2. Wash the cells with PBS and detach with 1 mL trypsin. Stop the reaction with 3-4 mL complete medium (+10% FBS). Aspirate up and down, and filter through a 40 um filter into sterile FACS tubes.
3. Count the cells. Calculate the total required number of cells and aliquot into 1.5 mL eppendorf tubes (or 15 mL tubes if more cells needed)
4. Spin down at RT in a benchtop centrifuge (3 min, 1200 rpm)
5. Remove the supernatant and resuspend the pelleted cells in the appropriate volume of PBS. Transfer to sterile Bijou tubes (for easier handling later)
6. Keep the cells on ice. Place the mice in the heating chamber.
7. Gently pipette the cell suspension up and down with a P1000 before each cage injection. Prepare the injections for all of the mice in the cage, aspirating around 50 uL extra. Just before the injection, mix the cells in the syringe by moving the plunger up and down. Adjust the volume, pick up the mouse, and inject. Repeat the process for each cage.
TITLE:     Direct ELISA for investigating the binding of recombinant or chemically-made Protein-AG  to immunoglobulins.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein AG (SpAG) is an immunoglobulin-binding protein that interacts with the Fc regions of many mammalian immunoglobulins [1].</div><div class = "text-block">References</div><div class = "text-block">1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175 </div></div>

[STEPS]
?. This ELISA is used to study the interaction of protein-AG (SpAG) with diverse immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins or 50 µl of any animal sera in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-protein-AG conjugate diluted 1:3000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 2 SD.
TITLE: Neuro-muscular Impairments in Prolapsed Lumbar Intervertebral Disc (PLID) according to Structural Diagnosis and Management (SDM)
AUTHORS: Md. Shahadat Hossain , K M Amran Hossain, Sapia Akter, Foisal Mohammad Mosiul Alom

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The study aims to determine the Neuro-muscular Impairments in Prolapsed Lumbar Intervertebral Disc (PLID) according to Structural Diagnosis and Management (SDM). Structural Diagnosis and Management (SDM) of musculoskeletal medicine focuses on a comprehensive approach to assess both the contractile and non-contractile structures to generate a hypothesis to solve the specific sources hindering normal activities in the lumbar spine. This has been developed through 15 years’ experience of Dr. M Shahadat Hossain treating diverse cases of musculoskeletal medicine. The SDM assessment directs conservative management procedure in a way of taking history, examination of arthokinemetic and osteokinemtic motions of lumbar spine, stretching the series of muscles in lumbopelvic and lower extremities with biomechanical rationale, strength test to relative prime, segmental and regional stabilizer, neurological examination to the biomechanical contributor, myotomes and isolated neural structures to generate a diagnosis. </div></div>

[STEPS]
?. [Observation and Validation]
Finalizing Protocol
?. [Observation and Validation]
Application for Ethical Approval
?. [Observation and Validation]
Planning and Preparation for Data Collection
?. [Observation and Validation]
Assessment and Documentation of PLID patients according to SDM
?. [Observation and Validation]
Data Input and Audit
?. [Observation and Validation]
Result preparation
?. [Observation and Validation]
Manuscript preparation
?. [Intervention ]
Designing intervention and prepare of experimental trials
TITLE: Nucleic acids extraction from single cell using MasterPure Complete DNA purification (Epicenter)
AUTHORS: Sarah Romac

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Radiolaria are protists which can't be cultivated. These microoganisms have to be isolated by single-cell for genetic identification and it can be difficult to get clean DNA.</div><div class = "text-block">Here we optimized a DNA extraction protocol from protist single-cell.</div><div class = "text-block">It works very well on single-cell Radiolaria, Foraminifers, but also Ciliates, Dinoflagelletes, Diatoms.</div></div>

[STEPS]
?. [1.    Cell Isolation]
Isolate individually protist cells (at least 50µm in length) using a glass bent micropipette under a binocular microscope.
?. [1.    Cell Isolation]
Wash each cell in three successive baths of 0.22µm-filtered and sterile seawater.
?. [1.    Cell Isolation]
Transfer subsequently cells in a 1.5mL sterile microtube.
?. [1.    Cell Isolation]
Add 30 μL of lysis buffer (Tissue and Cell Lysis Solution from MasterPureTM DNA and RNA Purification Kit, Epicenter) and store at -20°C.
?. [2.	Cell lysis]
Pellet cells by centrifugation (2 min at Vmax), throw the supernatant, let ~25-30 µL of liquid.
?. [2.	Cell lysis]
Dilute 1 µL of Proteinase K in 300 µL de lysis solution Tissue et Cellule for each sample. Vortex 10 sec for resuspending cells (facultative).
?. [2.	Cell lysis]
Add 300 µL of mix Proteinase K + lysis solution Tissue et Cellule in each sample. Vortex.
?. [2.	Cell lysis]
Incubate 15 min at 65°C , 1000 rpm. Put samples in ice 3-5 min.
?. [3.	Total nucleic acids precipitation]
Add 150 µL MPC reagent to 300 µL of lysed sample. Vortex.4-+ 500 µL Propan-2-ol. Mix per inversion.5-67-+ 500 µL d’EtOH 70%. Don’t vortex, mix gently the support. Spin 5min at Vmax ;at 12°C.8-Discard a maximum of supernatant with precaution, without touching the pellet.9-Let dry 5-10 min at RT.10-
?. [3.	Total nucleic acids precipitation]
Spin 10 min at 11 000 g, 12°C. If there is no pellet, add more 25 µL MPC buffer and spin again 10 min at 11 000 g, 12°C.
?. [3.	Total nucleic acids precipitation]
Transfer the supernatant ion a new clean microtube (1,5 mL), discard the pellet.(To keep the squeletton, keep the tube with the pellet, add 500 µL MilliQ Water and store at -20°C).
?. [3.	Total nucleic acids precipitation]
Add 500 µL of Isopropanol. Mix per inversion. Spin 10min at Vmax, 12°C.
?. [3.	Total nucleic acids precipitation]
Discard the supernatant with precaution, without touching the pellet.
?. [3.	Total nucleic acids precipitation]
Add 500 µL of Ethanol 70%. Don’t vortex, mix gently the support. Spin 5min at Vmax ;at 12°C.
?. [3.	Total nucleic acids precipitation]
Discard a maximum of supernatant with precaution, without touching the pellet.
?. [3.	Total nucleic acids precipitation]
Let dry 5-10 min at room temperature..
?. [3.	Total nucleic acids precipitation]
Elute in 25 µL of TE1x buffer.Vortex and spin shortly.Store at - 80°C.
TITLE: Oral Fat Tolerance Test (OFTT) in Human
AUTHORS: Francine dos Santos-Macedo, Dilliane da Paixão Rodrigues Almeida, Bianca Martins Gregório, Fernanda Amorim de Morais Nascimento Braga, Caroline Fernandes-Santos

[DESCRIPTION]
Following the consumption of a standardized high-fat meal, the oral fat tolerance test (OFTT) assesses the postprandial lipemic (PPL) response of blood triglycerides (TG). Blood samples are obtained four hours after meal ingestion, with a targeted TG level of ≤ 220 mg/dL considered within the normal range. An altered PPL response is defined by TG levels exceeding 220 mg/dL. The PPL is influenced by various factors, such as the quality and type of foods ingested in the diet, physical exercise, body metabolism, and genetics. The PPL response may predict the development of Atherosclerotic Cardiovascular Disease, thus serving as an early marker of the atherosclerotic process associated with metabolic disorders.

[BEFORE_START]
Depending on the blender available, preparing the volume of 4 meals at once is more practical because there are losses on the blender's wall. Participants must fast for 8 to 10 hours before blood collection. They should avoid consuming fatty foods and engaging in physical activity in the 72 hours before the test.

[STEPS]
SECTION: OFTT meal preparation (milkshake)
3. Weight the ingredients
SECTION: OFTT meal preparation (milkshake)
1. Identify the cup with the participant's name.
SECTION: OFTT meal preparation (milkshake)
2. Weigh the empty cup and record it (W1).
SECTION: OFTT meal preparation (milkshake)
4. Add the ingredients to the blender and mix.
SECTION: OFTT meal preparation (milkshake)
5. Distribute the milkshake into plastic cups.
SECTION: OFTT meal preparation (milkshake)
6. Weigh the whole cup and record it (W2).
SECTION: OFTT meal preparation (milkshake)
7. Place the lid and straw.
 


 


SECTION: Oral Fat Tolerance Test (OFTT)
8. Collect venous blood from the arm using a 5 mL syringe and reserve it in a serum separator tube.
SECTION: Oral Fat Tolerance Test (OFTT)
9. Offer the test meal to the study participant.
SECTION: Oral Fat Tolerance Test (OFTT)
10. Monitor the time it takes for the participant to consume the milkshake. They should consume as much as possible.
SECTION: Oral Fat Tolerance Test (OFTT)
11. After consumption is complete, weigh the cup (W3).
SECTION: Oral Fat Tolerance Test (OFTT)
12. Collect a new venous blood sample from the arm 4 hours after the start of meal ingestion.
SECTION: Calculating milkshake intake
13. Apply the following formula:
  

SECTION: Serum triglyceride assay
14. Centrifuge the blood at 2,000 x g for 10 minutes.
SECTION: Serum triglyceride assay
15. Transfer the serum to 2.0 mL microtubes.
SECTION: Serum triglyceride assay
16. Conduct the biochemical assay following the manufacturer's instructions.
TITLE: Thawing adherent cancer cell lines
AUTHORS: Emily Souster, Verity Goodwin, Charlotte Beaver, Adam Jackson, Fiona Behan, Rizwan Ansari, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP is for thawing an adherent cancer cell line from a frozen cryovial.</div><div class = "text-block">Process diagram:</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/b94e6458af3e4aa17dc6c162a5d28b87f529d83e9d05c0764f6eb3fffa72f21d/ccghbdsax.png" /></div></div>

[STEPS]
?. Remove the cryovial from liquid nitrogen storage and place on dry-ice for transfer to cell culture lab.
?. Take the cryovial from the dry-ice and hold in a   waterbath until thawed. Dry the cryovial thoroughly, spray with 70% ethanol and transfer to cell culture hood.
37 °C
Be careful here to avoid submerging the lid or rim of the cryovial in the waterbath.
?. Use a P1000 pipette to transfer the contents of the cryovial to the T75 flask containing  media.
18 mL
?. Use  complete culture media to wash the cryovial and add this to the flask.
1 mL
?. Place the flask in a  , 5% CO2 incubator. Agitate the flask gently back and forth and side to side to ensure even distribution of cells across the flask.
37 °C
?. After 24 hours, inspect adherence and confluency. Remove the media using a sterile aspirating pipette and replace with   complete culture media.
12 mL
TITLE: Rotarod (40 RPM and 20 RPM with docking) 
AUTHORS: daniel.dautan, Per Svenningsson

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

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

[STEPS]
1. Naive mice were placed in groups of 5 on the rotatord (Ugo Basile 47650) for 2-3 minutes for habituation. 
2. Following habituation, the rotarod program was initiated and lasted for a maximum of 2 minutes. 
4. The time to fall as defined with the moment the mice fall in the receptacle or the moment the mice turn around the rotor more than 5 consecutive times was selected.
6. The test was repeated 3 times with at least 6h between each trial.
5. The apparatus was thus cleaned thoroughly between each group with 50% ethanol followed by water.
3. Following habituation, the rotarod program was initiated for 40 RPM and lasted for a maximum of 2 minutes.
3. Following habituation, the rotarod program was initiated for 20 RPM on docking move (5 rotation by cycle) and lasted for a maximum of 2 minutes.
4. The time to fall as defined with the moment the mice fall in the receptacle or the moment the mice turn around the rotor more than 5 consecutive times was selected.
5. The apparatus was thus cleaned thoroughly between each group with 50% ethanol followed by water.
6. The test was repeated 3 times with at least 6h between each trial.
TITLE: Sephadex/sephacryl purification of AFLP products
AUTHORS: Michal Ronikier, Tomasz Suchan

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Preparation of 96x plates]
Put the Sephacryl bottle on the shaker for 5-10 minutes, adjust speed to 4-5 to make it liquid. For the last minute add also the Sephadex mixture bottle and decrease the speed to 3-4.
?. [Preparation of 96x plates]
Prepare a 1:1 (v/v) solution of pre-prepared 5% Sephadex G50 and Sephacryl S200 (35 ml of each) and mix well.
?. [Preparation of 96x plates]
Pipet 300 µl of the mixture to each well of the Millipore purification plate.NOTE: Use the Matrix electronic pipet >> Fill at 1250 µl, Disp at 300 µl).
?. [Preparation of 96x plates]
Place the Millipore plates on trash plates.
?. [Preparation of 96x plates]
Centrifugate 1 min at 2800 rpm.Purification of AFLP products
?. [Purification of AFLP products]
Match and place the prepared purification plates (Millipore plates with fresh Sephadex/Sephacryl layer prepared as described above) on new Eppendorf plates.describe the contents of the Eppendorf plate beforehand.double-check carefully the orientation of the two plates!fix the two plates additionally with small pieces of tape on both sides.
?. [Purification of AFLP products]
Pipet the whole volume of the selective PCR product onto the centre of Sephadex/Sephacryl membranes without touching the membranes (using multichannel pipette).
?. [Purification of AFLP products]
Centrifugate 2 min at 2800 rpm.
?. [Purification of AFLP products]
Remove the Millipore plate and use the filtrated PCR product in the Eppendorf plate for subsequent dilution of the selective PCR product.
?. [Purification of AFLP products]
Prepare the mix of 10 µl of Hi-Di formamide and 0.1 µl of Gene Scan-500 ROX size standard per sample and pipet 1-1.5 µl of the purified and diluted PCR product.
Millipore plates can be reused up to three times. Leave the Sephadex resin to dry in ambient temperature – it will contract and become easy to remove. Then, plates can be loaded with a new Sephadex/Sephacryl mixture.
TITLE: Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-Salmonella antibody in layer hen&#39;s egg yolks.
AUTHORS: Angel Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-</span><span style = "font-weight:bold;font-style:italic;">Salmonella</span><span style = "font-weight:bold;"> antibody in layer hens was a reproducible and feseable test used to meassure IgY development after vaccination. </span></div></div>

[STEPS]
?. U-shaped bottom's ninety-six well polystyrene microplate purchased at Sigma-Aldrich, St. Louis USA was incubated with (2 µg/well) of the LPS (Sigma –Aldrich) from Salmonella Typhimurium in coating buffer (overnight at 4 ºC.)
?. The microtiter plates was washed four times, with 10 % PBS-Tween-20.
?. The microplate was blocked with 3% non-fat milk in PBS (25 µl/well).
?. The microplate was incubated 1 hr at RT .
?. The microplate was washed four times.
?. Then a 50 µl aliquot of the egg yolk (Ig)Y solutions in a concentration of 1.25 mg/ml was added in triplicate. The IgY concentration was assessed by ELISA and sample were titrated with sample buffer until it got the expected IgY concentration.
?. After incubating for one hour at RT, the microplate was washed four times.
?. Fifty (50 µl) of the anti-IgY-HRP conjugate (Sigma-Aldrich) diluted to 1:30000 with conjugate diluent was  added into each well.
?. The microplate was incubated for 1 hr at RT.
?. Then, the microplate was washed four times.
?. Fifty (50 µl) of tetramethylbenzidine (TMB, Sigma-Aldrich) was added into each well.
?. The microplate was further incubated for 15 minutes in the dark.
?. Fifty (50 µl) 3M HCl was added to the microplate for stopping  the reaction.
?. After that, reaction  color development was measured with a microplate reader (Synergy™ Neo Hybrid Multi-Mode Microplate Reader).
?. The cut-off point was an OD of 0.51, and it was calculated from the XOD of the negative control times 3. This ELISA tested triplicates of a total of 90 IgY preparations.
TITLE: PCR Prep from cDNA for IVT Reactions
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes PCR1 and PCR2 reactions beginning with cDNA to generate transcript templates ready for IVT reaction.  This protocol is written for 192 (2x96) reactions but can also be used for fewer or more reactions by adjusting the amount of materials and equipment appropriately.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: Om-mCherry-hsp90
AUTHORS: Claudio Slamovits, Pia Elustondo, Ronie Haro, Susana sbreglia@dal.ca

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Map and sequence of the construct used to transform Oxyrrhis marina using electroporation. </div></div>

[STEPS]
?.