eamag/protocols-with-steps huggingface dataset:
TITLE: UV Crosslinking of Suspension Cells for eCLIP
AUTHORS: Eric Van Nostrand, Gene Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description for UV crosslinking of suspension cells to prepare them for eCLIP experiments.</div></div>

[STEPS]
?. [UV Crosslinking]
Aliquot at most 60×106 cells (re-suspended in 1x DPBS) in at least 3 mL total volume to a standard 10cm tissue culture grade plate.
?. [UV Crosslinking]
Place the above (plate plus ice or cooling block) into the UV cross-linker.  Note: Ensure the plate is leveled.  Remove tissue culture plate lid for cross-linking.
?. [UV Crosslinking]
After crosslinking is completed, transfer the cells to a 50 mL conical tube
?. [Wash Cells]
Resuspend the pellet(s) in 25 mL of 1x DPBS at room temperature.
?. [Wash Cells]
Spin down remaining sample in 50ml conical tube(s) at 200g for 5 minutes at room temperature.
?. [Wash Cells]
Count cell concentration (either with automated cell counter or hemocytometer)
?. [Wash Cells]
Aspirate supernatant
?. [UV Crosslinking]
Wash plate once with 7mL of 1x DPBS and add to the same 50 mL tube.
?. [UV Crosslinking]
Cross-link at 254-nm UV with an energy setting of 400 mJoules/cm2
?. [UV Crosslinking]
Gently resuspend until the sample is homogeneous
?. [UV Crosslinking]
Count cell concentration (either with automated cell counter or hemocytometer)
?. [UV Crosslinking]
Resuspend in the desired amount for flash freezing
?. [UV Crosslinking]
Aspirate and discard supernatant.
?. [UV Crosslinking]
Centrifuge the 50 ml conical tube at 200g for five minutes at room temperature.
?. [UV Crosslinking]
After frozen (at least thirty seconds), remove from the liquid nitrogen and store at -80°C.
?. [UV Crosslinking]
Aspirate the supernatant and freeze by submerging the epi-tubes completely in liquid nitrogen.
?. [UV Crosslinking]
Spin down at 200g for five minutes at room temperature.
?. [UV Crosslinking]
Transfer desired amount into 1.5 mL Eppendorf Safe-Lock Tubes (or equivalent)
?. [Prepare Suspension Cells]
Pool all cells per biosample (if multiple plates)
?. [Prepare Suspension Cells]
Aspirate spent media
?. [Prepare Suspension Cells]
Centrifuge at 200g for 5 minutes at room temperature
?. [Prepare Suspension Cells]
Transfer cells with media to 50mL conical tube(s)
?. [Wash Cells]
Resuspend cells to no more than 20×106 cells per mL
?. [UV Crosslinking]
Place the tissue culture plate on leveled ice or a cooling block pre-chilled to 4°C
TITLE: Immunofluorescent Staining of Fixed Mouse Brain Tissue Sections
AUTHORS: Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
This protocol describes steps for immunofluorescent staining of free floating fixed mouse brain tissue sections.

[STEPS]
1. Free floating sections in 12-well plates, all steps performed at room temperature.
1.1. 2 x 10min PBS washes.
1.2. 2 x 10min 0.2% PBS-Triton (PBS-T) washes.
1.3. Block 1hr in blocking buffer.
1.4. Incubate in primary antibody diluted in blocking buffer overnight.
1.5. 3 x 10min 0.2% PBS-T washes.
1.6. Incubate in secondary antibody diluted in blocking buffer for 2hrs.
1.7. 2 x 10min PBS-T washes.
1.8. 2 x 10min PBS washes.
1.9. Mount sections on glass slides, add mounting media and coverslip.
TITLE: Single cell RNA-seq and ATAC-seq protocol for PBMCs treated with LPS
AUTHORS: Francesca Luca

[DESCRIPTION]
Single cell RNA-seq and ATAC-seq protocol for PBMCs treated with LPS

[STEPS]
1. Cell Processing – day 1
2. Transfer the selected cryovials from liquid nitrogen into dry ice then store in -80C. The day before seeding the plates (Day 1)
3. Day 1:
4. 1. Thaw one vial of PBMCs in the water bath. Transfer immediately to 6mL of room temperature SCAIP media and mix gently using a wide-bore tip. Rinse the cryovial with an additional 1 ml of media.
5. 2. Count cells on Countess: 10ul cells + 10ul Trypan Blue, Check for viability, and record on the Cell counting sheet page
6. 3. Repeat for 11 additional samples.
7. 4. Centrifuge cells at 400xg for 10 minutes.
8. 5. Resuspend cells to 2x106 cells/mL in a culture medium.
9. 6. Plate 4 wells x 100ul in round-bottom 96-well plates using a wide-bore tip (4wells/individual). Each individual is a separate column.
10. 7. Incubate in SCAIP Media overnight at 37C and 5% CO2
11. 8. For each sample, spin the remaining Cells and freeze the pellet for DNA (-80C freezer).
12. Cell Processing – Day 2
13. Protocol:
14. 1. Prepare treatments (LPS, PHA, Dex, EtOH) – see Treatments page
15. 2. Add 2ul of treatments to their respective wells (multi-channel).
16. 3. Incubate for 6 hrs.
17. On ice:
18. 4. Take the plate out of the incubator and pool across rows/individuals (4 pools of 12 individuals):
19. * Use multi-channel to gently mix the media using a wide-bore tip and transfer column1(treatment plate) to column 1 of the deep-well plate. Then repeat to transfer columns 2-12 (treatment plate) to column 1 of the deep-well plate. Next, wash columns 1-12 in the treatment plate with 100ul cold PBS then pool to column 2 of the deep-well plate.
20. 5. Pool each row into a 5 ml tube using a wide-bore tip (4 tubes total).
21. 6. Centrifuge @300rcf, 5 min, 4oC as per the 10X SC Protocol.
22. 7. Remove the supernatant, wash with 5 ml ice-cold PBS+0.04% BSA, and centrifuge again.
23. 8. Resuspend in 1ml ice-cold PBS+ 0.04% BSA.
24. 9. If significant amounts of cell clumps or debris are observed, gently mix cells by pipetting up and down 10 – 15 times and filter cells using a Flowmi Cell Strainer (40 µm).
25. 10. Count cells on the countess, check viability, and record.
26. 9. Make sure cell concentration is within the target range of 0.7 M/ml – 1.2 M/ml (aim to capture 25k cells by loading 60K). Adjust if needed. Ideally, viability should be 90% and above acceptable above 80%. Proceed with the 10x Genomics® Single Cell Protocols
TITLE: Stereotaxic rat brain surgery for Substantia Nigra targeting
AUTHORS: miquel.vila

[DESCRIPTION]
Stereotaxic rat brain surgery 

[STEPS]
1. Anesthetize rat with isofluorane (5% induction phase, 2% maintenance phase)
2. Weight rat & shave the head
3. Inject meloxicam (0.5 mg/mL) and Baytril (5 mg/mL)
4. Place rat head gently with ears bar onto the stereotaxic equipment
5. Scalpel + Scalpel holder = incision middle of rat head
6. Turn vacuum on 
7. Place 4 hemostats in each extremity of the incision (opening in square shape)
8. Add hydrogen peroxide. Clean. Add saline . Clean.
9. Remove remaining blood with cotton swab
10. Remove tissue of the skull with the not-cutting side of scalpel (scratch in order to see bregma and lambda as better as possible, can bleed for a prolonged time, apply saline as necessary)
11. Check skull horizontality: place drill (adjust with the yellow tool) + take DV in lambda and bregma by placing the drill on the cranium (neither above nor pressing it). If not leveled (+/- 0.1 between Bregma and Lambda), adjust as necessary 
12. Drill very slowly. Regularly check the depth of craniotomy.
13. Inject vector solutions using a 10 µL Hamilton syringe fitted with a glass capillary
14. Go to Bregma with the Hamilton syringe
15. Calculate SN coordinates: AP (5.2), ML (2.0), DV (7.6)
16. Perform infusion at 0.4 µL/min and let the needle in place for 4 minutes
17. Seal craniotomoy with bonewax using a small spatula
18. Sew the incision 3-5x
19. Add skin glue to incision site
TITLE: U Michigan - Spot urine collection
AUTHORS: Jeff Hodgin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Collection of urine from experimental animals is a basic requirement to detect albumin/creatinine ratio for glomerular nephropathy. This is a non-invasive, simple, quick, and inexpensive method for collecting spot urine from mouse when a small volume of urine (10-500ul) sample is required.</div></div>

[STEPS]
?. Create 6 evenly spaced holes in the lid of the container – 3 on top and 3 at bottom using an electric drill using 1/8” drill bit. Also drill 2 holes each on four sides of the container. Using a metal cutter cut out a 14.5 cm by 9.5 cm piece of stainless steel wire mesh to create a base for mouse that will fit inside the lean mouse cage. Then bend all four sides of the wire mesh using a flyer to create 1 cm perpendicular bent on each sides. Insert the wire mesh base inside the container with the side bents pointing downward. The base should have a snug-fit inside the container about 1” above the bottom. Create similar way for obese mouse cage. Wire mesh need to be washed and autoclaved before using.
?. It is important to acclimatize the mouse in the metabolic cage for about half hour to one hour every day for 3 consecutive days before starting urine collection.
?. Prepare appropriate number of urine collecting tubes by labeling a tube with identification number for each animal.
?. To maintain SPF housing, place cages in an operational laminar flood hood.
?. Place the appropriate mouse in the metabolic cage and replace the cage lid.  Be certain the lid is secure. Put one mouse per box and label the top of the box with the mouse number.
?. Place the mouse cage card in front of metabolic cage. Make sure mouse cage card has all the necessary information such as mouse identification number, mouse protocol number, emergency contact number, Principal Investigator name, date, and time period of 4 hour urine collection, also “DO NOT FEED” sticker (Provided by Lab Animal Facility)
?. Because mice need to be left without food or water for 4 hours, an exception must be completed.
?. Collect urine for 4hour period without food and water access.
?. Check on mice every hour to monitor mouse health conditions.
?. After 4hour remove the mouse from the metabolic cage and replace in the original housing cage and carefully collect the urine.
?. Collect urine using a pipette (P200 is usually best) and place in a 1.5 mL micro centrifuge tube.
?. Spin urine at 2000 rpm for 5 min. Remove supernatant and place in a clean 1.5 mL tube.
?. Store urine samples at 4ºC until ready for assay in short period and at -20ºC or -80ºC for a longer period.
?. Clean metabolic cage with appropriate disinfectants.  Hot, soapy water may be used on the acrylic parts.  All cage parts should be sprayed with a bactericidal, virucidal, and fungicidal (e.g., Clidox® or Spor-Klenz ®) and wiped down.  Dry each part and reassemble
TITLE: Luminescent Conjugated Oligothiophenes (LCO) Staining
AUTHORS: Toby J Curless, Hemanth Ramesh Nelvagal, Zane Jaunmuktane

[DESCRIPTION]
The protocol describes Luminescent Conjugated Oligothiophenes staining on brain sections.




[STEPS]
SECTION: Preparation
1. Generate tissue sections using standard microtome sectioning protocols. 

SECTION: Deparaffinisation and LCO Staining
3. 
 


De-paraffinise sections by 5 min washes in xylene (x2), 5 min 100 % ethanol (x2) and 5 min in DEPC H2O (x1).

SECTION: Preparation
2. Heat tissue dry tissue sections for 60 min at 60 °C



SECTION: Deparaffinisation and LCO Staining
4. Circle tissue on slide using a hydrophobic ImmEdgeTM PAP pen (Vector laboratories) to contain
solution.

SECTION: Deparaffinisation and LCO Staining
5. Prepare 1:500 pFTAA solution (diluted in PBS). All steps from here should be performed away from light.

SECTION: Deparaffinisation and LCO Staining
6. Pipette pFTAA solution onto tissue and incubate at room temperature in a humidified chamber for 30 minmins.

SECTION: Deparaffinisation and LCO Staining
7. Wash slides in PBS 3x.
SECTION: Deparaffinisation and LCO Staining
8. Coverslip slides using HydromountTM aqueous non-fluorescing mounting media (National Diagnostics).
TITLE: Total DNA extraction from plant tissue using CTAB method
AUTHORS: Robert Auber

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

[STEPS]
?. Using a pre-chilled mortar and pestle, grind frozen plant tissue (1-5g grams) into a fine powder
?. Add 500 ul of pre-heated 2 x CTAB buffer (with beta-mercaptoethanol added) to the pelleted cells. Vortex and re-suspend by gently pipetting up and down.
60 °C
?. Incubate at 60 C for 30 min. Periodically, mix gently during the incubation.
60 °C
?. Add an equal volume of chloroform:isoamyl alcohol (24:1), mix very well at RT for 5 min to generate a monophasic solution.
?. Centrifuge for 10 min in 5,000 x g
?. Transfer aqueous phase (upper layer) to new tube.
?. (Optional) Repeat chloroform:isoamyl alcohol extraction (steps 3 and 4) until the interface is clean.
?. Add 2/3 volumes of isopropanol, mix gently for 5 min at RT.
?. Centrifuge for 15 min at 10,000 x g. Discard supernatant.
?. Add 500 uL  of 70% ethanol to the DNA pellet, and briefly vortex to dislodge the pellet.
?. Air dry pellet for 30 min. Resuspend in 100ul of TE Buffer.
?. Add 1 ug/mL DNase-free RNase (final concentration) and incubate at 37C for 30 min.
?. Extract the DNA once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Centrifuge at 10,000x g for 10 min. Transfer the aqueous phase to a clean tube.
?. Precipitate DNA with 1/3 volume of 7.5 M ammonium acetate and 2.5 volumes of 100% ethanol.
?. Centrifuge at 10,000x g for 10 min, wash with 70% ethanol, and air dry for 30 min at RT. Resuspend in 50 ul of TE Buffer. Allow the DNA to dissolve overnight at 4C
TITLE: Protein gel sample preparation 
AUTHORS: Cleo B.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sample preparation for tricine SDS-page gels and polyacrylamide SDS-page gels. </div></div>

[STEPS]
?. [sample preparation tricine SDS page]
If you are unsure about the amount of protein present in your sample, load multiple concentrations Add beta-mercaptoethanol to tricine sample buffer until a final concentration of 2% Dilute 1 part sample with 1 part sample buffer
?. [sample preparation  SDS page]
If you are unsure about the amount of protein present in your sample, load multiple concentrations Add  of beta-mercaptoethanol per  of 4x laemmli sample buffer Dilute 3 parts sample with 1 part sample buffer
100 µl
900 µl
?. Spin samples down ~ 10 sec Place samples at  for 5 min Load samples to gel
95 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>For cDNA synthesis and for amplify larger fragments of YFV genome. The PCR target is of the CprM/envelope region of YFV genome described by Jorge and colleagues (2017).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Power Analysis Apo Field work
AUTHORS: Tom Little, Nick Colegrave

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We used </span><span style = "font-weight:bold;">Power analyses</span><span> to clarify our ability to detect the effects of stressors as main effects, or as interactions, with these sample sizes. We observed in ref</span><a href="https://www.zotero.org/google-docs/?HKigKm" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">12</span></a><span> that food supplementation similar to our proposed methods can cause a shift in, for example, body weight of ~ 0.75 standard deviations from the mean, and we make the assumption that we will achieve a similar effect. We constructed a linear model with chronological age as a covariate with a normal distribution and a relationship to the response variable with a slope of 0.9. We then added sex and both food and drug treatments (full model: response = age + food*drug*sex) to ask if treatment effects could be detected at the p<0.05 threshold in at least 80% of the cases during 1x105 iterations (80% is a standard threshold in power analyses</span><a href="https://www.zotero.org/google-docs/?hj8uuJ" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">55</span></a><span>). From this, we are confident we will have the power to detect main effects within a single season. For example, where food supplementation drives an increase in the response across both sexes or across both drug and control individuals, power was >0.9, i.e there is a 90% probability of finding a significant effect. If, however, food drove the expected response in only one sex or only in drug treated individuals, our power to detect this interaction was ~0.56, below the required power threshold. Our power to detect interaction effects rises to >0.8 when the larger sample size of both field seasons are incorporated. We also observed in ref</span><a href="https://www.zotero.org/google-docs/?02fme2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">12</span></a><span> that ‘grid’ effects average ~0.5SD, and so performed further analysis with six ‘grids’, where treatments were nested within ‘grid’ for the statistical model. This analysis confirmed that the proposed sample size for one field season allows for the detection of main effects, but power to detect interaction effects requires both field seasons. </span></div></div>

[STEPS]
TITLE: In vitro depalmitoylation assay
AUTHORS: wusj, schekman

[DESCRIPTION]
This protocol describes the in vitro depalmitoylation assay of DNAJC5

[STEPS]
SECTION: In vitro depalmitoylation assay
1. HEK293T, MDA-MB-231, or Hela cells were transfected with plasmids encoding DNAJC5 by lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
SECTION: In vitro depalmitoylation assay
2. Cellular membranes were prepared as described in "Membrane and cytosol fractionation protocol"
SECTION: In vitro depalmitoylation assay
3. For chemical deplamitoylation, the membranes were resuspended and incubated with 0.5 M hydroxylamine (pH 7.2) or 0.5 M Tris (pH 7.2, control) at Room temperature   in the presence of a cocktail of protease inhibitors

SECTION: In vitro depalmitoylation assay
4. The mobility of DNAJC5 was examined by SDS-PAGE followed by immunoblot.
TITLE: sohbet
AUTHORS: Sohbet Odalari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Sıcak </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">sohbet odaları</span></a><span>, yetişkin sohbet ve </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">chat</span></a><span> imkanını mobil cihazlarınızla deneme imkanı sunan </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">sohbet</span></a><span> ve chat siteleri.</span></div></div>

[STEPS]
?. sohbet
?.
TITLE:       ELISA for quantification of IL-35 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Regulatory B cells mainly secrete IL-35. One of the primary functions of this interleukin is its involvement in lymphocyte differentiation. It exhibits an immune-suppressive effect. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-35 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-35 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-35 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-35 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-35 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-35 standard dilutions and the human IL-35 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Microfluidic Chip Production
AUTHORS: Suresh Poovathingal, Florian De Rop, Stein Aerts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for producing microfluidic chips used in HyDrop experiment.</div></div>

[STEPS]
?. [Microfluidic Chip Preparation]
Microfluidic device fabricationGuide to making microfluidic devices for bead production. The same method is used for the droplet encapsulation chips.
?. [Microfluidic Chip Preparation]
Bake 4h @ 100 °C and store the finished chips with tape covering the inlets.
?. [Microfluidic Chip Preparation]
Check if bubbles are gone, pour into PMMA holder on wafer or PU mold. Pour leftover PDMS mix into a cell culture plate to create a PDMS mat. Bake in over for 4 hours (or at the shortest 2 hours) @ 80 °C
?. [Microfluidic Chip Preparation]
Remove PDMS slab from mold and tape over features to prevent dust accumulation Puncture slabs with 1 mm biopsy needles and PDMS mat to prevent tip bluntingA quality of life tip: when you puncture the slabs, do so at a slight angle so that the tube that you will connect through that hole will face slightly outward, away from the centre of the tip. This ensure that the tube will not block your microscope's light source.
?. [Microfluidic Chip Preparation]
Put glass slide into 50 mL falcon and add 1 mL of acetone.Shake wellWipe glass plate dry with dust-free cloth
?. [Microfluidic Chip Preparation]
Put glass plate with rough yellow edge on top on the glass plasmabonder platePut chip with features facing up on the glass plasmabonder platePut plasmabonder vacuum valve in the metering positionVacuum glass + PDMS chip in plasma bonder until the pressure is stable under 1 mmHgPut plasma on HIGH for 30 secondsTurn off plasma bonder and take out the plateSlowly press PDMS slab on glass plate. Press down well using flat object.Check for delamination
?. [Microfluidic Chip Preparation]
Flow 10 uL of silane in HFE (2%) through the channels Wet all channels quicklyBlow out all oil in channels using the air gun. Any residual HFE will evaporate and leave a concentrated mess on the surface of the chip.