eamag/protocols-with-steps huggingface dataset:
TITLE: Western Blot (semi-dry)
AUTHORS: Anna Behle, Anika Wiegard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for semi-dry Western Blot</div></div>

[STEPS]
?. [Western Blot]
Equilibrate gel in Cathode buffer for 30 minutes (no shaking)
?. [Western Blot]
Activate PCDF-Membrane for ca. 20 seconds in MeOH, incubate for 2 minutes in dH2O (no shaking)
?. [Western Blot]
Equilibrate PCDF-Membrane for 5 minutes in Anode buffer
?. [Western Blot]
Wet two Blotting papers (thickness 1 mm each) in Anode buffer and place on Anode
?. [Western Blot]
Put PCDF-membrane on top
?. [Western Blot]
Put Gel on top
?. [Western Blot]
Wet two blotting papers (thickness 1 mm each) in Cathode buffer and place on gel.
?. [Western Blot]
Cut membranes and papers as exactly as possible to gel size (Careful! Gel gains some size while equilibrating) and place each component exactly on top of eachother. After each step consider eliminating air bubbles by rolling them out with a glass rod with buffer.
?. [Western Blot]
Run blot at 1,5 mA/cm2 for 90 minutes (depending on the size of the protein, the blotting duration will have to be adjusted)
?. [Immunodetection]
Block membrane with TBST + 5% milk powder for 45 minutes
?. [Immunodetection]
Wash membrane with TBST and primary antibody for at least 1h at RT or 4°C over night. Anti-gfp antibody will be diluted 1:5000 in TBST, use as little volume as possible to preserve the expensive antibody.
?. [Immunodetection]
Wash for 10 minutes in TBST. Repeat 3 times.
?. [Immunodetection]
Add secondary antibody and incubate at least 45 minutes at RT or at 4°C over night. Swivel gently.
?. [Immunodetection]
Wash for 10 minutes in TBST. Repeat 3 times.
?. [Immunodetection]
Mix Bio-Rad Clarity Western 1:1 (ca. 1 ml per membrane). Let membrane drain, evenly add developer to membrane (with 1ml tip). Spread between film (remove bubbles), incubate for 1 minutes and detect.
?. [Immunodetection]
Blots; Chemi or Chemi-high Sensitivity, Signal Accumulation mode, transmission light for ladder detection
?. [Immunodetection]
Don't forget to take a picture of membrane for merge!
?. [Stripping]
Wash blot in TBS
?. [Stripping]
immerse in stripping buffer
?. [Stripping]
incubate at RT for 5-15 minutes (Under the hood!)
?. [Stripping]
remove and wash again in TBS
?. [Stripping]
reprobe!
TITLE: 20X Anatomic and Layer Identification for Biocytin Filled Cells
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To characterize the orientation, laminar position, and anatomical location of in vitro single cells reconstructed in mouse and human brain slices, a 3-step process is used. Briefly, 20X brightfield and/or fluorescent images of DAPI (4',6-diamidino-2-phenylindole) are annotated to determine layer position and brain region, of biocytin-filled cells.</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: LEGACY01: DATA AND SAMPLE SHARING
AUTHORS: Katrina M Pollock, Calliope Dendrou

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

[GUIDELINES]
DATA AND SAMPLE SHARING

The protocol will be published and made open access during the study. 

During the study, data will be shared through controlled access using the following principles:

No data should be released that would compromise an ongoing trial or study. 

There must be a strong scientific or other legitimate rationale for the data to be used for the requested purpose. 

Investigators have a period of exclusivity in which to pursue their aims with the data before data are made available to other researchers. 

Adequate resources must be available in order to comply in a timely manner or at all, and the scientific aims of the study must justify the use of such resources. 

Data exchange complies with Information Governance and Data Security Policies in all the relevant countries.

Researchers wishing to access study data should contact the chief investigator in the first instance. 

New algorithms or methods of value to the broader community will be shared online via the GitHub and/or protocols.io websites.

At the end of the study, single-cell data will be archived and made open access as part of the Human Cell Atlas data portal (https://data.humancellatlas.org/), and other data (e.g., imaging, bulk RNA sequencing, genotyping, flow cytometric, and serological data) will be made open access through deposition in the European Genome-phenome Archive (EGA; https//ega-archive.org), Cytobank (https://mrc.cytobank.org/cytobank/) and Zenodo (https://zenodo.org), for example. Meta-data deposited along with the sequencing data will be MINSEQE-compliant. 

[STEPS]
TITLE: Culturing 3T3 Cells for Validating GEARBOCS 2.0 Constructs 
AUTHORS: Justin T Savage

[DESCRIPTION]
This protocol is for culturing Cas9 NIH/3T3 cells for the validation of GEARBOCS 2.0 and similar constructs for use in in vivo assays.

[STEPS]
1. Validation by indel sequencing in immortalized murine cell lines:
4. Seed Cas9 NIH/3T3 cells at 250k per well of a 6-well plate (if starting from frozen cells, you. will need to passage at least once prior to transfection. So thaw to 10cm dish, then passage to 6-well)
5. 24 hours after seeding, transfect Cas9 NIH/3T3 cells with GBOCS plasmid:
6. For each gene of interest, combine the following components in a 1.5 mL tube:
7. Mix by vortexing
8. Add 6 uL of Xtreme Gene
9. Pipette up and down rapidly. Do not touch pipette tip to the wall of the tube (Xtreme gene is sticky and extremely finicky. You want to minimize contact between XG and plastic surfaces)
10. Mix tube contents by gently flicking
11. Incubate 30 min at room temperature.
12. Add 200 uL of optimem/XG/DNA mixture per well in drop-wise swirlfashion.
13. Return cells to incubator for 48 hours
14. 48 hours after transfection, collect cells and isolate genomic DNA for Sanger sequencing:
15. Place 6-well plate on ice
16. Aspirate media and wash once with 1 mL of ice-cold PBS per well
17. Aspirate PBS wash and add fresh 1 mL cold PBS for collection
18. Use rubber scraper to collect cells into 1.5 mL tube and keep on ice
19. Use Qiagen DNeasy kit to isolate genomic DNA
6.1. 200 uL optimum
1 uL of GBOCS2.0 (at 1ug/uL)
2. Prepare HEK293 cell media
3. 1. DMEM 450ml
2. Fetal Bovine Serum 50ml
3. L-Glutamine, 2 mM, 5ml
4. Sodium Pyruvate, 1 mM, 5ml
5. Pen-Strep, 100 U/mL, 5ml
TITLE: DNA extraction protocol for snake genomic sequencing by using AxyPrepTM Multisource Genomic DNA Miniprep Kit
AUTHORS: Boyang Liu, Liangyu Cui, Zhangwen Deng, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
Genomic DNA was extracted from the muscle of a snake using the AxyPrepTM Multisource Genomic DNA Miniprep Kit (Axygen, China), with some modifications from the kit instructions as outlined in this protocol. The extracted DNA was then suitable for WGS sequencing.

[STEPS]
1. The following protocol is a modified DNA extraction protocol using the AxyPrepTM Multisource Genomic DNA Miniprep Kit. 

Approximately 10mg of snake tissue was minced into small pieces using 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 the DNA.
1 min Room temperature
TITLE: Copy of Copy of Copy of          ELISA for quantification of human C9 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C9 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C9 present in the serum or plasma 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-C9 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-C9 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 C9 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 C9 standard dilutions and the human C9 sample concentration is determined.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol described a easy procedure to purify anti-sera in small scale batch. Protein A or protein G beads can be used depending on the immunoglobulin types.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Overall protocol for MicroPOTS LCMS top down proteomics of kidney tissue sections
AUTHORS: Mowei Zhou, James M Fulcher, Yen-Chen Liao, Ljiljana.PasaTolic

[DESCRIPTION]
This is the overall workflow for LCMS top down proteomics of kidney functional units from tissue sections using the MicroPOTS platform. The expected outcomes are proteoform identification and quantitation values from selected tissue functional units.

[STEPS]
SECTION: Tissue collection
1. The tissue sections were prepared and shipped from Vanderbilt-TMC following the protocol below:

 

SECTION: Sample preparation
2. Functional units (glomerulus, medullary, tubule) were dissected and collected into the microPOTS platform using the method below:
 

SECTION: Data Acquisition
3. The samples were analyzed by LCMS top down proteomics as described below:
 

SECTION: Data Analysis
4. LCMS datasets were analyzed for proteoform identification and quantitation. The final results are reported.
TITLE: Evaluation of the Antibacterial efficacy of Allium Sativum Gel as Intracanal Medicament Versus Calcium Hydroxide Paste Against Enterococcas faccalis in Single Rooted Teeth (A Comparative In-Vitro Study)
AUTHORS: Rania Ahmed Ahmed ELnaa

[DESCRIPTION]
Objectives
The aim of this study is to evaluate the antimicrobial efficiency of garlic extract as an intracanal medicament in comparison to calcium hydroxide paste against Enterococcus faecalis viability developed in single rooted root canals by bacterial DNA extraction method using real time PCR.
Research Question
Will application of intracanal medicament of garlic extract differ from calcium hydroxide paste in reduction of E. Faecalis count when used in single rooted teeth?
 Null Hypothesis
There is no difference between the antimicrobial efficacy of garlic extract and calcium hydroxide paste in reduction of E. faecalis count in single rooted teeth.
PICO elements 
P: Population
Single rooted human freshly extracted teeth with single canals.
I: Intervention
Garlic extract used as intracanal medication.
C: Control
Calcium hydroxide paste used as intracanal medication.
O: Outcome
Bacterial load reduction determined by using real time PCR technique after root canal preparation.
Time of sampling 
Sample 1 (S1): after inoculation of root
canals& before instrumentation.
Sample 2 (S2): after instrumentation.
 Sample 3 (S3): after intracanal medicament placement for 1 week. 
Sample 4 (S4): after intracanal medicament placement for 2 weeks.

[STEPS]
TITLE: Amplification of  DNA  
AUTHORS: Yuichiroh  Ikagawa

[DESCRIPTION]
Our project aims to quantify human fatigue through the quantification of the biomarker human herpesvirus6 using Cas12a, a protein that is fundamental and essential to our project. We ordered the synthesis of composite parts with affinity tags attached to Cas12a, but the sequence was too long to be synthesized as a single of dsDNA. To overcome this problem, we split the parts into three fragments and synthesized them. Here, an adaptor sequence was added to the 5' end by PCR to allow for later assembly.



[STEPS]
1. Thaw the DNA solution at room temperature, and KAPA HiFi HotStart ReadyMix (x2) on ice. 
2. Vortex the reagents, then centrifuge them briefly in a microcentrifuge.
3. Mix the reagents according to the composition in the table below.
 

 

4. For each template DNA, select the temperature setting of the thermal cycler listed in the table below.
4.1. Vectors(pSB1C3 and pSB1A3)

 

4.2. Cas12a fragments(1-3)
TITLE: Swabbing scats for DNA sampling - field protocol
AUTHORS: Anna MacDonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to swab mammal scats in the field, for DNA sampling. Swabs are air-dryed for storage in the field and transport to the laboratory.</div></div>

[STEPS]
?. Before touching the scat:photograph the scatassign a sample numberrecord GPS coordinatesrecord any other required field data
?. Swab the scat before otherwise moving or disturbing it.
Wear clean gloves for every sample. Change gloves as often as needed to avoid contamination.
?. Select a clean, unused swab and check that the paper seal is undamaged to ensure the swab is sterile (discard the swab if the seal is damaged).
?. Record the sample number and all other details on the swab tube label. Ensure the writing is clear and legible and will not smudge or wash off easily.
?. Twist the swab to break the seal and carefully remove the swab from its case. Take care not to touch any part of the swab that was inside the collection tube and do not allow the swab to touch anything else that may contaminate it.
?. If the scat still retains obvious moisture, there is no need to pre-moisten the swab. Proceed to step 8.
?. If the scat is dry (older scats), pre-moisten the swab before use: Immediately after opening the swab, open a 2ml tube of 1 x PBS Briefly dip the swab into the PBS solution – you just need to moisten the outside of the swab, it should not be         saturated or soaked for a long time Immediately close the tube of PBS to avoid contaminating it Briefly dip the swab into the PBS solution – you just need to moisten the outside of the swab, it should not be         saturated or soaked for a long time Immediately close the tube of PBS to avoid contaminating it
ONLY dip clean swabs into the PBS solution – if the swab has touched ANYTHING (your hands, the ground, the scat...) do not dip it into the tube! Otherwise you risk cross-contamination of other swabs later on.
?. Swab the scat comprehensively by rolling the tip of the swab across the surface of the scat several times. Make sure you sample from each scat pellet and from the ends and middle of the scat.
NB see this EcoGene video on YouTube for a demonstration of a similar procedure (swabbing starts at 1 min 38 secs): https://www.youtube.com/watch?v=A7WFnoIttUA
?. Briefly allow the swab to air dry – take care not to touch the swab against anything that may contaminate it while it dries.
?. Place the swab back into its container and ensure it has properly closed. Swab containers can be stored in a box or bag during the field trip. They should be kept dry and out of direct sunlight. Store used swabs separately from unused swabs.
?. In the lab, extract DNA from swabs as soon as possible, or else store swabs in dry conditions below 4°C (freeze if possible) to inhibit growth of micro-organisms.
TITLE: Fixation and Immunostaining protocol
AUTHORS: gustavo.parfitt Parfitt, Gist Croft

[DESCRIPTION]
Fixation and Immunostaining protocol for tissue and cell culture

[STEPS]
1. Prepare 1x Fix by dilution from 2x Fix (see Description)
2. Gently aspirate culture supernatant, wash gently with PBS, and replace with cold Fix buffer
3. Incubate 30min @ 4° C or on ice
4. Aspirate fix and wash gently 3x with PBS (incubate 1-2min each)
5. Aspirate  and add Quench to permeabilize cells/tissue, at least 15 min room temperature (RT). Note: this and all steps in humidified chamber (HC). Can store samples for weeks/months at 4 degrees
6. Aspirate and add Block buffer, 30 min RT
7. Incubate with primary antibodies cocktail (1-2 hrs RT or 4°C overnight (ON)
8. Wash 3x  5min (wash buffer)
9. Prepare secondary antibodies in Wash buffer (1:500-1:1000), filter, light protect (LP).Note: This and all subsequent steps LP
10. Incubate with secondary antibodies 1hr RT, light protected (LP)
11. Wash 3x 5min
12. Incubate with 1x DAPI in Wash buffer 5-10 min
13. Wash 1x
14. Rinse very briefly with filtered H2O, 2x
15. Remove excess water, do not dry samples
16. Add Fluoromount-G (~20ul/well or ~100ul/slide) and apply No 1.5H coverslip (slowly lower, no bubbles) or other mountant
17. Dry ON, LP, RT
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol will introduce a workflow for quality control and pre-processing of metagenomic sequence reads using FastQC for visualization and FastX Toolkit for editing the fastq files. </p>

[GUIDELINES]
<p><a href="http://hannonlab.cshl.edu/fastx_toolkit/index.html" target="_blank">FastX Toolkit Documentation</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Synthesis of derivatization reagent TAHS (N,N,N-trimethylamonioanilyl N-hydroxysuccinimidyl carbamate iodide)
AUTHORS: Manuel Liebeke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">TAHS was synthetized according to the protocol previously described with minor modifications. TAHS is used as derivatization reagent for amino acids. The purpose is to generate enough TAHS for on-tissue derivatisation to enhance signals during MALDI-MSI of amino acids from tissue sections.</div></div>

[STEPS]
?. 6.0 g (23.4 mmol) DSC was dissolved in 250 mL of dry ACN to which 3.0 g (22.0 mmol) DPD in 250 mL dry ACN was added dropwise over a period of 45 min and stirred (Room temperature)DSC Molecular Weight 256.17 C9H8N2O7DPD Molecular Weight 136.19 (CH3)2NC6H4NH2
[DSC]
[DPD]
[ACN]
?. The product was concentrated using rotary evaporation and resuspended in 5 mL ACN and filtered through Whatman filter paper No.2 Qualitative.The product should be checked by mass-spectrometry and NMR for puritym/z 278.1141 [M +H]+, molecular formula, C13H16N3O4
?. The filtrate was concentrated and redissolved in 10 mL 4:1 ACN:DCM after which 0.4 mL iodomethane (8 equiv.) was added and left to react overnight at room temperature.
?. The reaction mixture was filtered to afford approx. 0.3 g of TAHS (6.55 mmol, 29.8% yield over 2 steps).
?. The product TAHS should be checked by mass-spectrometry and NMR for puritym/z 292.1297 [M]+,molecular formula, C14H18N3O4 (Δ -2.9 ppm).
TITLE: Inducing proteostasis stress using G-TPP
AUTHORS: Louise Uoselis

[DESCRIPTION]
Inducing proteostasis stress in HeLa cells with G-TPP to analyse proteostasis protection and repair.

[STEPS]
SECTION: Day 1
1. Seed cells in standard growth media, aiming for a density of ~80-90% the following day. 
SECTION: Day 3
4. Harvest the G-TPP treated samples and DMSO control samples after the desired incubation period (eg. 2 – 12 h).
SECTION: Day 2
2. Feed cells for 60 min prior to starting treatment by aspirating the media from each well and replacing with a standard volume of growth media (eg. Feed with 2 mL of media if using a 6 well plate).

SECTION: Day 4
7. Harvest the 24 h recovery (24 h R) time point, at 1440 min after the washout step (steps 5 – 6) was performed.

SECTION: Day 2
3. Aspirate the media from each well/plate, and replace with standard growth media that has had G-TPP added at a final concentration of 9 micromolar (µM) (for the DMSO control, add the same volume of DMSO as you added G-TPP to each well/plate).  

SECTION: Day 3
5. For samples to be analysed followed after G-TPP removal, aspirate the treatment media from each sample, wash each sample 3x in an ample amount of room temperature PBS (eg. 3 mL per well for a 6 well plate).
SECTION: Day 3
6. After 3x PBS washes, add an appropriate volume of standard growth media to each well/plate (eg. 2 mL of standard growth media per well for a 6 well plate). 
SECTION: Day 4
8. Replace the media on the plate for the 48 hour recovery (48 h R) samples with an appropriate volume of standard growth media (eg. 2 mL of standard growth media per well for a 6 well plate).   
SECTION: Day 5
9. Harvest the 48 h R samples, 1440 min after the samples had fresh standard growth media added (step 8).
TITLE: Expression and purification protocol of GST-NDP52
AUTHORS: Chunmei  Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the expression and purification of GST-NDP52.</div></div>

[STEPS]
?. [Protein expression]
Transform the E.Coli BL21DE3 cells with plasmid encoding for GST-NDP52 and plate them on Amp plate.
?. [Protein expression]
Carry out protein expression in  medium, induce with  IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at .
1.5 L
18 °C
?. [Protein expression]
Harvest the cells by spinning at  for  at  and stock at  until purification.
Centrifuge: 4500 34
4 °C
-80 °C
?. [Protein purification]
Follow the GST batch purification by Size Exclusion Chromatography.
?. [Protein purification]
Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at  at  for
Centrifuge: 16000 33
4 °C
?. [Protein purification]
Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at  with gentle shaking for , apply to a gravity column, and wash extensively with Wash Buffer.
4 °C
?. [Protein purification]
Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer at .
4 °C
?. [Protein purification]
Pool the peak fractions containing pure protein, snap-frozen in liquid nitrogen, and store at .
-80 °C