eamag/protocols-with-steps huggingface dataset:
TITLE: TS Spurrs - Cell Monolayer on Thermanox coverslips (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
Processing a cell monolayer on Thermanox coverslips to Spurrs resin block for EM.

[GUIDELINES]
All step times are the minimum time required. Longer steps are acceptable.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:


SECTION: Conventional - with reduced times for monlayer thickness
2. 2.5% % volume     in 0.1 Molarity (M) Sorenson's phosphate buffer pH 07.2 300 mosmol/kg20 min.

SECTION: Conventional - with reduced times for monlayer thickness
3. Wash buffer 5 min 

SECTION: Conventional - with reduced times for monlayer thickness
4. 
1 Mass / % volume   in buffer30 min 

SECTION: Conventional - with reduced times for monlayer thickness
5. 70 % volume    10 min 

6. 95 % volume   5 min 

7. 100 % volume   5 min 

8. 100 % volume    5 min 

9. 100 % volume Spurrs:100 % volume    in 1:1 ratio30 min 

10. 100 % volume Spurrs30 min 

11. 100 % volume Spurrs30 min 

12. Oven polymerise 65 °C
TITLE: Recharge PM Weight Loss: Reviews, Side Effects &amp; Precautions!
AUTHORS: brettlee  

[DESCRIPTION]
Product Name – Recharge PM
Composition –Natural Organic compound
Side-Effects–NA
Availability– Online
Rating –★★★★★
Official Website– Recharge PM.com

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[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study is to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis – both with respect to expression and function.</p>
<p>Synovial fluid, blood and synovial samples will be obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue will analysed by immunohistochemistry and flow cytometry. SF and blood will be analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration will be determined using a Boyden chamber. Appropriate statistical tests will be applied for comparisons.</p>

[STEPS]
TITLE: Transferrin uptake assay to measure Clathrin-mediated endocytosis in HIPCS derived neurons.
AUTHORS: Sakthi Kumar

[DESCRIPTION]
This protocol is used to measure Transferrin uptake as a way to investigate Clathrin-mediated endocytosis in HIPCS derived neurons, or other cell types.

[STEPS]
SECTION: Reagents needed:
1. 1. Human Transferrin conjugated with Alexa Fluor 647 (Thermo Fisher, Catalog no: T23366).
Note: Transferrin conjugate is also available with Alexa Fluor 488, Alexa Fluor 546. The user may want to use a specific conjugate based on the need of their experiments. We have optimized this protocol for Transferrin conjugated with Alexa Fluor 647.
2. Basal Neuronal media- No added supplements like (N2, B27, NEAA, Glutamax and growth factors). 
Note: The user may use their desired neuronal medium, but they have to make sure that the media does not contain any Transferrin.
SECTION: Preparation of Transferrin aliquots:
2. Dissolve 5mg of Transferrin in 1 mL of Sterile PBS.
Make the aliquots of desired volume and store at 2-8 C. 
Protect them from light.
Do not freeze the aliquots.
For long term storage, add Sodium Azide.
SECTION: Experimental Protocol
3. Warm the basal neuronal media to 37 °C.
Add warmed basal media to a new 24 well plate (500uls/ well needed) in the tissue culture hood.
Transfer the coverslip with cells (neurons) into the well and proceed to starvation step.
Starvation: starve the cells by keeping the plate in the humidified incubator (37 °C, 5% CO2) up to 60 min
After 30 to 60 minutes, remove the plate from the incubator and add Transferrin (10ug/ml) to the cells and keep the plate in the incubator for 1 min.
After 1 minute, remove Transferrin and add 4% PFA for fixation.




SECTION: Experimental Protocol
3.1. PFA fixation:
After adding 4% PFA wait for 15 min for fixation.
Then wash 3 times with PBS.
Add Hoechst and incubate for 10 min.
Then wash 3 times with PBS and mount the coverslips on slides with Fluoromount G.
Image the same day or keep the slides at 4 °C if imaging the next day.


SECTION: Experimental Protocol
3.2. Quantification:
Use ImageJ based program to quantify Transferrin Fluorescence intensity. 
SECTION: Notes:
4. Cell types tested:
We have tested Transferrin uptake in multiple cell types generated from H9 Embryonic Stem Cells.
Cortical neurons.
Midbrain Dopaminergic neurons.
MGE derived GABA interneurons.
For the assay, we have used mature neurons from the above cell types (4 weeks after plating the progenitors).

Depending on the experiments, the user may want to test different concentrations of Transferrin and different time points of incubation. Also, the user may try pulse chase step to visualize recycling endosomes. For our experiments, we focused only on the initial uptake through Clathrin mediated endocytosis.
TITLE: Researcher led sample preparation for LC-MS using the BioMS research core facility
AUTHORS: ronan.ocualain

[DESCRIPTION]
This is a collection of protocols that covers the processing of most biological samples for proteomics, from collection of sample, up to data acquisition using LC-MS  

[BEFORE_START]
This method is suitable for the preparation of a wide variety of sample types for LC-MS analysis. It is assumed that you have already had a discussion about your work before attempting the steps described here. If not, contact us at bioms@manchester.ac.uk. 

To get you started, we will provide you with Covaris lysis tubes for each sample that you wish to process, at cost.

[GUIDELINES]
This method is suitable for the preparation of a wide variety of sample types for LC-MS analysis. It is assumed that you have already had a discussion about your work before attempting the steps described here. If not, contact us at bioms@manchester.ac.uk for a chat. 

[STEPS]
TITLE: Specificity index (pSI) calculation 
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Specificity index (pSI) calculation from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. To calculate the specificity index of genes detected within TRAP/RiboTag datasets of dopaminergic neurons and other cell types within midbrain, pSI (v1.1) was used with default settings22,24,38–40,73. Genes were considered significantly specifically expressed with an FDR-adjusted P value < 0.01.
TITLE: Constructs and generation of stable cell lines
AUTHORS: michela.deleidi

[DESCRIPTION]
Protocol used to generate stable Flp-In T-REx-HEK 293 cell lines expressing WT or mutant GCase (E326K or L444P) as
a V5-FLAG-tagged protein using a tetracycline-inducible system.


[STEPS]
1. Constructs were purchased from IDT (gBlocks Gene Fragments) and subcloned into pcDNA5/frt/to (Thermo Fisher Scientific, # V652020).
2. For the generation of V5-FLAG-GCase lines, the tag was positioned at the N-terminus, three aa after the cleavage site of the leader sequence. These three amino acids are repeated after the tag to ensure that the GBA1 sequence is intact.
3. To ensure proper cleavage, the V5-FLAG tag was inserted 12 bp after the cleavage site, and this 9 bp were repeated after the tag. To avoid interference with proper protein folding, we employed the short V5 sequence (IPNPLLGLD).
4. Site-directed mutagenesis was performed according to the manufacturer’s protocol (Agilent, QuickChange II XL), and base pair exchange was confirmed by Sanger sequencing.
5.  Flp-In 293 T-Rex cells(Thermo Fisher Scientific, #R78007) were grown in media composed of Dulbecco’s modified Eagle’s medium (DMEM, Sigma–Aldrich), 10% fetal bovine serum
(Gibco), 1% GlutaMax (Gibco) supplemented with 100 μg/ml Zeocin
(InvivoGen) and 15μg/ml blasticidin (InvivoGen).
6. Inducible Flp-In T-Rex 293 cells were generated according to the manufacturer’s protocol
(Thermo Fisher Scientific). 
7. The selection was performed with DMEM supplemented with 15μg/ml blasticidin and 100
μg/ml hygromycin B Gold (InvivoGen) 48 h after transfection and continued until the
expression of the gene of interest was induced by treating the cells with 50 ng/ml doxycycline hyclate (Sigma–Aldrich) for 48 h.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol to transiently transfect <em>Capsaspora owczarzaki</em>, a unicellular eukaryote</p>

[BEFORE_START]
<p><strong>Transfection Reagents preparation</strong></p>
<p> </p>
<p><strong>Growth medium (for 1 L)</strong>: 10 g Peptone (BD, #211677), 10 g Yeast Extract (BD, #212750), 1 g<br />Yeast nucleic acid (Ribonucleic Acid, Type VI from Torula Yeast) (Sigma, #R-6625), 15 mg Folic acid<br />(Sigma, #F8758) in 880 mL distilled water. Autoclave for 15 min at 121ºC. Cool down and aseptically<br />add 0.4 mL of Hemin stock solution* (Sigma, #H9039), 20 mL Buffer solution** and 100 mL of heat-<br />inactivated Fetal Bovine Serum (Sigma, #F9665-100ml). Filter-sterilise through 0.22 µm and store at<br />4ºC.</p>
<p><strong>*Hemin stock solution (for 200 mL)</strong>: 400 mg NaOH in 200 mL dH 2 O. Add 500 mg of Hemin and<br />autoclave 20 min at 121ºC. Store at 4ºC protected from the light.</p>
<p><strong>**Buffer solution (for 1 L)</strong>: 18.1 g KH 2 PO 4 (Sigma, #P5655), 25 g Na 2 HPO 4 (Sigma, #S5136) in 1 L<br />distilled water. Adjust final pH to 6.5 with HCl 37% and filter-sterilise through 0.22 µm. Store at 4ºC.<br /><strong>Transfection medium (for 1 L)</strong>: 10 g Peptone, 15 mg Folic Acid in 990 mL distilled water. Autoclave<br />for 20 min at 121ºC. Aseptically add 10 mL HEPES 1 M (Sigma, #H4034) to a final concentration of<br />10 mM and 2.1 g Bis-Tris methane (Sigma, #B9754) final concentration 0.21% w/w. adjust pH to 7.1<br />with NaOH, filter-sterilise through 0.22 µm and store at 4ºC.<br /><strong>2X HBS (for 250 mL)</strong>: Dissolve 4 g NaCl (Sigma, #S3014), 0.18 g KCl (Sigma, #P9541), 0.05 g<br />Na 2 HPO 4 (Sigma, #S5136), 2.5 g HEPES and 0.5 g D-glucose (Sigma, #G8270) in autoclaved distilled<br />water. Adjust pH to 7.1 with NaOH. Filter-sterilise through 0.22 µm, flash-freeze with liquid Nitrogen<br />and store at -80ºC.<br /><strong>1.25M CaCl 2 (for 10 mL)</strong>: 1.84 g CaCl 2 (Sigma, #C1016) in 10 mL autoclaved distilled water. Filter-<br />sterilise through 0.22 µm, flash-freeze with liquid Nitrogen and store at -80ºC.<br /><strong>10% glycerol (for 4 mL)</strong>: 0.8 mL of filter-sterilised 50% (v/v) glycerol (Sigma, #G7757) in 1.2 mL<br />autoclaved distilled water and 2 mL 2X HBS. Filter-sterilise through 0.22 µm, flash-freeze with liquid<br />Nitrogen and store at -80ºC.</p>

[GUIDELINES]
<p>-This protocol is set up for transfecting <em>Capsaspora</em> in 12 wells/plate. All volumes are defined per well.</p>
<p>-Prepare reagents in advance</p>
<p>-When replicates are to be done, a larger DNA mix can be set up</p>

[STEPS]
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TITLE: PacBio Isoseq samples preparation
AUTHORS: anita.adami

[DESCRIPTION]
This protocol described how to prepare samples for PacBio Isoseq

[STEPS]
SECTION: RNA extraction
1. The total RNA was obtained from brain tissue samples using miRNA Easy Mini Kit (Qiagen). The manufacturer's instructions were followed step-by-step.
SECTION: Samples QC evaluation
3. At the Nation Genomics Infrastructure of Sweden, input QC of samples was performed on the Agilent Bioanalyzer instrument, using the Eukaryote Total RNA Nano kit to evaluate RIN and concentration.  
SECTION: RNA extraction
2. RNA samples were subsequently put on dry ice and shipped to National Genomics Infrastructure of Sweden. 
SECTION: Libraries preparation
4. The sample libraries were prepared as described in "Procedure & Checklist – Iso-Seq™ Express Template Preparation for Sequel® and Sequel II Systems" (PN 101-763-800 Version 02 (October 2019)) using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module, the Iso-Seq Express Oligo Kit, ProNex beads (Promega) and the SMRTbell Express Template Prep Kit 2.0. 
SECTION: Libraries preparation
5. 300 ng of total RNA was used for cDNA Synthesis followed by 12 + 3 cycles of cDNA Amplification. 
SECTION: Libraries preparation
6. In the purification step of amplified cDNA, the standard workflow was applied (sample is composed primarily of transcripts centered around 2 kb). 
SECTION: Libraries preparation
7. After purification, the amplified cDNA went into SMRTbell library construction. 
SECTION: Libraries preparation
8. Quality control of the SMRTbell libraries was performed with the Qubit dsDNA HS kit and the Agilent Bioanalyzer High Sensitivity kit.
SECTION: Libraries preparation
9. Primer annealing and polymerase binding was performed using the Sequel II binding kit 2.0.
SECTION: PacBio Isoseq sequencing
10. Finally, the samples were sequenced on Sequel II and Sequel IIe System using Sequel® II Sequencing Plate 2.0, On-Plate Loading Concentration of 110 pM, movie time 24 hours and pre-extension time 2 hours.
TITLE: RNA extraction from Escherichia col
AUTHORS: Alice Pawlowski, Lutz Berwanger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA extraction from E. coli cells based on the method described by Chomczynski and Sacchi, 1987</div></div>

[STEPS]
?. [Cell prepartion]
centrifuge for 5 min at 4°C and 14000 x gdiscard the supernatant and resuspend the pellet in 1 ml NucleoZOL (Macherey and Nagel), place on dry iceproceed to next step or store cells at -20 or -80 °C
4 °C
?. [Cell prepartion]
Wear safety gear
?. [RNA-isolation]
incubate the sample at 65 °C and 250 rpm (Thermomixer) for 10 minmix with 400 μl Chloroform/Isoamylalcohol (Roti®-C/I) by inverting for 10 s
65 °C
?. [RNA-isolation]
centrifuge at 4°C for 10 min at 14000 x gtransfer aqueous phase to a new reaction tube, work on icemix with 450 μl Penol/Chloroform/Isoamylalcohol (Roti®-Aqua-P/C/I)centrifuge at 4°C for 10 min at 14000 x gtransfer aqueous phase to a new reaction tube and add 1 Vol. icecold Isopropanol + 20 μl 3 M Na-Acetat (pH 5.2) and mix
[2nd centrifugation step]
[1st centrifugation step]
4 °C
Wear safety gear!
?. [RNA-isolation]
?. [RNA-isolation]
centrifuge at 4°C for 30 min at 14000 x gremove the supernatant (take care of the RNA-pellet) and add 350 μl of icecold 75% etahnol
4 °C
?. [RNA-isolation]
4 °C
?. [RNA-isolation]
add 350 μl of icecold 75% etahnol centrifuge for 5 min at 4°C and 14000g
4 °C
?. [RNA-isolation]
remove the supernatant and dry the pellet at room temperature for ca.15 minresuspend the pellet in 30 μl Molecular Biology Grade Water or TE-buffer (10 mM Tris/HCL pH 8.0 , 1 mM EDTA)
TITLE: 7T MRI Protocol for response to Respiratory-gated Auricular Vagal Afferent Nerve Stimulation
AUTHORS: Vitaly Napadow, Roberta Sclocco, Vitaly Napadow, Roberta Sclocco

[DESCRIPTION]
This is the 7T MRI protocol for a study of the subject to Respiratory-gated Auricular Vagal Afferent Nerve Stimulation in  the area of the auricular concha. Scan types include: multi-echo MPRAGE and 6 GRE BOLD scans for the stimulation conditions.

[STEPS]
TITLE: Single-Nuclei Isolation From Snap Frozen Axolotl Brain
AUTHORS: Ashley  Maynard, Fides  Zenk

[DESCRIPTION]
This protocol enables isolation of single nuclei from frozen pallium microdissections and whole pallium dissections (from axolotl) for the purpose of generating single-nuclei gene-expression libraries following a modified protocol from 10x (Demonstrated protocol CG000365, Rev B). In brief, we prepared and precooled wash and lysis buffers (see Materials). Lysis buffer was added to the sample and dissociated via short pulses with an electric grinder. The pestle of the grinder was washed with a wash buffer before centrifugation. Supernatant was removed and the pellet gently washed. After a final centrifugation the supernatant was removed and the pellet was resuspended in PBS + BSA. Resulting nuclei were then assessed (count and viability) using Trypan Blue assay, counted using the automated cell counter Countess (Thermo Fisher). The resulting nuclei are then ready for downstream analysis, including but not limited to 10x Genomics gene expression (single-nuclei RNA sequencing).

[STEPS]
SECTION: Nuclei isolation
2. Use pre-cooled buffers and storeon ice, perform isolation steps on ice, use pre-cooled micro-centrifuge at 4 °C.

SECTION: Nuclei isolation
3. Put tissue in cold 1.5 mLtube

SECTION: Nuclei isolation
4. Add 50 µLof lysis buffer

SECTION: Nuclei isolation
5. Using an electric grinder. Grind the tissue for 10 s  (or 2-5 pulses depending on if the tissue persists) in the tube. Rinse the pestle with 150 µL wash buffer.

SECTION: Nuclei isolation
6. Optional: Check an aliquot of the nuclei on the Evos or at the Nikon
SECTION: Nuclei isolation
7. Spin down 5 min at 500 x g (at 4 °C)

SECTION: Nuclei isolation
8. Optional: Keep the supernatant and check an aliquot on the Evos or at the Nikon
SECTION: Nuclei isolation
9. Wash the pellet with 200 µL of wash buffer (do not disturb the pellet for optimal recovery)

SECTION: Nuclei isolation
10. Spin down at 500 x g for 5 min (at 4 °C)

SECTION: Nuclei isolation
11. Resuspend the pellet in 50 µL of wash/resuspension buffer

SECTION: Nuclei isolation
12. Count with typan blue (5 µLsample + 5 µLtrypan)

SECTION: Prepare Buffers
1. Prepare the buffers as described in the Materials section. Store buffers at 4 °C or on ice.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Today, we are going to get you set up for your class project. You will need a class directory for putting all of your files. Also, you will need to download the data for your project (see the list of SRR numbers in D2L).</p>

[STEPS]
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TITLE: Macherey-Nagel Nucleospin 96 Food protocol for bee pollen
AUTHORS: Lauren  Ponisio, Jocelyn Zorn

[DESCRIPTION]
Macherey-Nagel Nucleospin 96 Food protocol for bee pollen

[STEPS]
1. UV sterilize supplies for 2 96 well plates worth of extractions: 4 50mL centrifuge tubes, 2 15mL centrifuge tubes, zirconia beads, 2 96 deep well plates and clear strip caps, 2 s-blocks, 2 96 well elution plates, 14 1000uL tip boxes, 2 200uL tip boxes, 2 10uL tip boxes, 6 reagent troughs, and 6 96 well microplates 
2. Aliquot   into sterile centrifuge tube and warm in 65 °C water bath. You will need to aliquot 30 mL per 1/2 plate (48 samples)

3. Add 540 µL   to each warmed 30 mL 
  aliquot and invert gently to mix

4. Add 560 µL   +   solution to each sample
 


5. Place in tissue lyser and run at 10 Hz for 1 min 

 

 

6. Centrifuge 3220 x g, 1 min 


 

7. Using sterile tweezers, remove bee leg, rinse with 200 proof ethanol, and place leg into labeled sterile microcentrifuge tube. Sterilize tweezers between each use with flame. Leave leg tubes open in fume hood until remaining ethanol has evaporated, then store at -80 °C 
 


8. Add ~100 µL zirconia beads to each pollen sample

9. Place in tissue lyser and run at 24 Hz for 90 s , rotate plates 180 degrees, and lyse again at24 Hzfor  90 s 

 

10. Place in 65 °C water bath for 30 min 

 

11. Centrifuge 3220 x g, 35 min 

12. Transfer 300 µL of supernatant into 96 deep well plate
 


13. Add 300 µL (or equal volume)   
and 300 µL (or equal volume) 200 proof ethanol
 


 

14. Vortex samples until thoroughly combined
15. Centrifuge 1500 x g for 30 s 

 


16. Place food binding plate onto s-block and transfer sample to food binding plate. Seal with gas-permeable foil
17. Centrifuge 3220 x g, 9 min , discard flowthrough

18. While centrifuging, aliquot out 12 mL per plate of   and place in 70 °C water bath

19. Add 500 µL   and seal
 


20. Centrifuge 3220 x g, 4 min , discard flowthrough

21. Add 900 µL   , do not seal

22. Centrifuge 3220 x g, 20 min , discard flowthrough

23. Incubate binding plate  37 °C  for 30 min 

24. Place binding plate over elution block. Add 100 µL pre-heated   directly onto center of each binding plate filter membrane

25. Incubate room temperature 5 min 

26. Centrifuge 3220 x g, 4 min 

27. Aliquot entirety of product into 3 96 well microplates per DNA plate
28. Store at -20 °C (short term) to -80 °C  (long term)
TITLE: Purifying DNA from an Agarose Gel
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for purifying DNA from an agarose gel. To see the full abstract and additional resources, please visit </span><a href="https://www.addgene.org/protocols/gel-purification" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/gel-purification/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Follow the agarose gel electrophoresis protocol with the following amendments:  Gel purification is most efficient with lower % agarose gels, so you will want to stay in the 0.7-0.8% range if possible.You will want nice crisp bands. This can be achieved by using a wider gel comb and running the gel at a lower voltage.You will want to have enough space around each band to cut without having DNA in other lanes contaminating your sample. To accomplish this, it is best to skip lanes between samples and between the ladder and nearest sample. To minimize the risk of DNA damage, it is best to limit the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the gel before cutting out the bands and you will want to use long-wavelength UV for as short a time as possible to get the bands cut out.
?. Once you have run your gel, move it to an open UV box.
be sure to wear proper UV protection - especially for your eyes!, remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
Notes:To protect the UV box, it is a good idea to place the gel on a glass plate if available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from being cut by the razor blade. Try to get as little excess gel around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important during the DNA purification step, as many kits cannot handle more than a certain total volume of gel per reaction.
?. Place the gel in a labeled microfuge tube.
?. Using a scale, weigh the tube with the gel fragment after zeroing the scale with an empty tube. Alternatively, you can just subtract the weight of the empty tube from the weight of the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
?. Finally, you will want to isolate the DNA from the gel. This is most commonly done with a commercial gel purification kit, such as the QIAquick Gel Extraction Kit. Always follow the manufacturer's instructions.
It is usually important to determine the concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified DNA. Find more information about DNA quantification here.