eamag/protocols-with-steps huggingface dataset:
TITLE: Ex vivo mouse kidney slice experiment 
AUTHORS: Wakana Shoda, Naohiro Nomura, Fumiaki Ando, Hideaki Tagashira, Takahiro Iwamoto, Akihito Ohta, Kiyoshi Isobe, Takayasu Mori, Koichiro Susa, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was used in a manuscript "Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against hyperkalemia.</div></div>

[STEPS]
?. Make an incision then cut the skin from the bottom to the top of the mice followed the abdominal line with the round side of the scissors.
?. Mice were deep anesthetized.
?. Cut the lateral skin to give free access to the abdomen.
?. With your finger, displace the intestine to the right, who should see the kidney.
?. Clip the sternum with the curve forceps, cut the ribs, do not cut the lungs, give access to the heart by moving the forceps. The heart should beat.
?. Pick the needle (22 gauge) through the right ventricle.
?. Cut the right auriula and perfused at 17 ml/min with Hank's balanced salt solution (HBSS) pH 7.4 at 37°C equilibrated with 5%CO2/95%O2
?. Perfuse, until the kidneys are clear of blood, the kidneys appear green-white.
?. Immediately, they were removed. Take out the capsule, cut the kidney in pieces of 3 to 5 mm thick.
?. Put these pieces in bubbling buffer at room temperature. Start immediately to cut thinner slices (approximately 0.5 mm) using a micro slicer (Natume Seisakusho CO., Ltd, Tokyo, Japan).Cut from the cortex to the papilla in order to avoid kidney damages.
?. The slices were incubated at room temperature for 20 min in equilibrated HBSS for recovery before treatment. The tissue slice should always be in bubbling solution.
?. Hold the tube in the 20 ml scintillation vial . The vial should contain 10 ml of drug solution (3 ml is the minimum).
?. The slices will be treated with drugs. Treatment should be in bubbling solution at room temperature.
?. After treatment, the slices were snap frozen in liquid nitrogen.
TITLE: PNA Synthesis
AUTHORS: Cathy  Miller

[DESCRIPTION]
Peptide nucleic acid is one of the more important oligonucleotide mimics. Although PNA has a significant change in structure relative to oligonucleotides, the binding between PNA and complementary nucleic acids still follows the principle of base complementary pairing, it even has higher affinity than natural nucleotides, and has stronger resistance to degradation by nucleases and proteolytic enzymes. Researchers have designed and synthesized PNA with various structures to optimize its biological stability and availability, target binding characteristics, and pharmacokinetic characteristics.

We provide customers with various types of PNA conjugates and tailor-made PNA products for customer projects to achieve scientific research purposes. Our product analysis service will help our customers perform product analysis without worrying about poor product quality.

[STEPS]
TITLE: Preparation and transformation of electrocompetent cells
AUTHORS: Andreas Sagen

[DESCRIPTION]
A protocol for electroporation of E. coli. Other bacteria may work, with optimization of transformation buffer and settings.

[STEPS]
SECTION: Preparation of GYT medium
1. In a sterile flask, add 400 mL distilled water
SECTION: Preparation of cells
4. Inoculate 500 mL of prewarmed LB medium from 25 mL overnight E. coli culture. Incubate at 37 °C and 300 rpm. Measure OD every 20 minutes, until OD600=0.4

 


SECTION: Transformation
13. Pre-chill cuvettes on ice for 5 min, and pre-heat LB plates with an appropriate selection agent and SOC medium at 37 °C for 60 min
SECTION: Preparation of GYT medium
2. Measure and add 0.6 g yeast extract, 1.25 g tryptone, and 50 mL glycerol

Materials:
 
 
 
SECTION: Preparation of cells
7. Centrifuge culture at 1000 rcf, 20 min, 4 °C, and resuspend pellet in 100 mL ice-cold 10% glycerol
SECTION: Preparation of cells
6. Centrifuge culture at 1000 rcf, 15 min, 4 °C, and resuspend pellet in 200 mL ice-cold water

 

SECTION: Preparation of cells
5. Transfer the culture to appropriate centrifugation containers, and cool on ice for 30 min
SECTION: Transformation
17. Plate an appropriate amount culture on selection agar plates
SECTION: Transformation
16. Flush 1 mL pre-heated S. O. C. medium, then transfer to a culture tube and recover at 37 °C and 200 rpm shaking for 60 min
SECTION: Transformation
15. Incubate plasmid-suspension mix for 1 minute, then perform electroporation with optimized settings
SECTION: Preparation of GYT medium
3. Filter sterilize through a 0.22-µm filter, and store in aliquots at 4 °C
SECTION: Preparation of cells
10. Measure OD600 of a 1:100 dilution of the cell suspension, and dilute the cells to between 2-3  × 1010 cells/mL

 

SECTION: Preparation of cells
9. Centrifuge culture at 1000 rcf, 20 min, 4 °C, and resuspend pellet in 2 mL ice-cold GYT medium

 

SECTION: Preparation of cells
8. Centrifuge culture at 1000 rcf, 20 min, 4 °C, and resuspend pellet in 25 mL ice-cold 10% glycerol
SECTION: Preparation of cells
11. Transfer 40 µl of the suspension to an ice-chilled electroporation cuvette and test whether arching occurs when an electrical discharge is applied. If so, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low
SECTION: Preparation of cells
12. Dispense 40 µL aliquots of the cell suspension into sterile, ice-cold microfuge , drop into a bath of liquid nitrogen, and transfer to a -80 °C freezer
SECTION: Transformation
14. Mix an appropriate amount plasmid to an aliquot of electrocompetent cells and transfer to a pre-chilled cuvette
TITLE: Imaging Mass Cytometry Modality Overview 
AUTHORS: Michelle Daniel, Marda Jorgensen

[STEPS]
?. Process donor organs into samples for analysis. Lymph Node: dx.doi.org/10.17504/protocols.io.bbgnijvehttps://drive.google.com/drive/my-driveThymus: dx.doi.org/10.17504/protocols.io.bbgmiju6Spleen: dx.doi.org/10.17504/protocols.io.bc3kiykw
?. Register organ donor, organs received,  and common coordinate region information in the HuBMAP UUID generator at https://uuid.hubmapconsortium.org
?. Prepare paraffin blocks from tissue samples. Cassette Processing: dx.doi.org/10.17504/protocols.io.8nzhvf6Paraffin Embedding: dx.doi.org/10.17504/protocols.io.bam9ic96
?. Package and send paraffin blocks to the Bodenmiller lab: dx.doi.org/10.17504/protocols.io.bf5njq5e
?. Conjugate primary antibodies using the Maxpar Antibody Labeling Kit: dx.doi.org/10.17504/protocols.io.bf5jjq4n
?. Cut and mount paraffin sections for staining and analysis.Sectioning: dx.doi.org/10.17504/protocols.io.bfz4jp8w
?. Antibody staining of paraffin sections for Imaging Mass Cytometry: dx.doi.org/10.17504/protocols.io.bfz8jp9w
?. Prepare a slide to use for compensation: dx.doi.org/10.17504/protocols.io.bf2djqa6
?. Tune the Image Mass Cytometer for acquisition: dx.doi.org/10.17504/protocols.io.bf2gjqbw
?. Data Acquisition by Image Mass Cytometry: dx.doi.org/10.17504/protocols.io.bf2ijqce
?. Compensation Slide Acquistion: dx.doi.org/10.17504/protocols.io.bf2jjqcn
?. Load  Imaging Mass Cytometry Datasets onto Hipergator:
?. Prepare and upload data to the HuBMAP HIVE  Globus landing pad using Hipergator.
?. Image Processing and Segmentation: https://github.com/BodenmillerGroup/ImcSegmentationPipeline
TITLE: Keto Tone Reviews: (2020 Update) SCAM or Legit Product, Where to Buy?
AUTHORS: healthshead  

[DESCRIPTION]
You want to get a natural, safe solution that really works. If you're like me then you know how hard it can be to find an all-in-one keto supplement that actually keeps your body in ketosis. That's why I'm excited about Keto Tone! You can listen and watch the video below - or scroll down to read more.

[STEPS]
TITLE: Effectiveness of exercise therapy and self-management education to improve physical activity levels in patients with acute exacerbations of chronic  obstructive pulmonary disease: protocol of a systematic review and meta-analysis
AUTHORS: Koki  Yamamoto, Mami Takayama, Tadayoshi Nonoyama, Yusuke Kon, Yoshiki  Saimon, Takashi  Kitagawa

[DESCRIPTION]
We aimed to evaluate the effectiveness of exercise therapy and self-management education in improving physical activity levels (PA) in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and to provide future research directions. We plan to include all randomized controlled trials (RCTs) of exercise therapy and self-management education or either intervention alone designed to improve PA in patients with AECOPD. We will determine the differential effects of exercise therapy and self-management education interventions on daily steps, moderate to vigorous activity time (MVPA), and sedentary equivalent time (SB) (P: AECOPD, I: 1. exercise therapy, 2. self-management education, 3. exercise therapy and self-management education, C: usual care, O: steps per day, MVPA, SB). In this review, we plan to use five databases. Two reviewers will screen articles to identify studies for inclusion.For risk of bias assessment, we will use Risk of Bias 2 tool.

[STEPS]
TITLE: Strike one hundred to educate one: measuring efficiency of collective sanctions experimentally
AUTHORS: Philipp CHAPKOVSKIY

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this paper, we test if sanctions applied to an entire group for the free-riding of one of its members can increase the level of cooperation within that group. To measure the efficiency of such collective sanctions, we conducted a lab experiment based on a standard public good game. The results show that overall, collective sanctions are inefficient. Moreover, when subjects are able to punish their peers, the level of cooperation is lower in the regime of collective sanctions than under individual sanctions. Both outcomes can be explained by a general disapproval of the collective responsibility for an individual fault: in the post-experimental survey, an absolute majority evaluated such regimes as unfair. But although collective sanctions are not an effective means to boost group compliance, there are nevertheless two insights to be gained here. First, there are differences across genders. Under collective sanctions, males' level of compliance is substantially higher than under individual sanctions while the opposite is true for females. Second, there were intriguing differences in outcomes between the different regime types. Under collective sanctions, a person who is caught tends to comply in the future, at least in the short term. In contrast, under individual sanctions, an individual wrongdoer decreases his or her level of compliance in the next period.</div></div>

[STEPS]
TITLE: Mitochondrial Genome Assembly and Annotation
AUTHORS: zehnpjr

[DESCRIPTION]
This is a step by step protocol for assembling invertebrate mitochondrial genomes, annotating the genomes , publishing the genomes and then step by step protocol for making a phylogenetic tree using the data from the mitochondrial genomes 

[STEPS]
SECTION: Novoplasty v4.3.1
1. After QC, we ran our illumina short reads through a program called Novoplasty v.4.3.1. for mitochondrial genome assembly. 
 
Next you want to upload a seed and a reference file. The seed is where you would want the assembler to start assembling the program. I used the COI of an already published mitochondrial genome. Next you will want to upload the reference genome. I used the already published mitochondrial genome of the sea spider Achelia bituberculata. 
Then you want to create a config.txt file for your assembly.  It should loo like this: 

Project:
-----------------------
Project name= A_M
Type= mito
Genome Range= 12000-22000
K-mer = 33
Max memory=
Extended log= 0
Save assembled reads= no
Seed Input= /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/Seed.fasta
Extend seed directly= no
Reference sequence= /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/reference.fasta
Variance detection=
Chloroplast sequence=


Dataset 1:
-----------------------
Read Length = 151
Insert size = 300
Platform= illumina
Single/Paired = PE
Combined reads=
Forward reads = /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/M_R1.fastq
Reverse reads = /mnt/home/zehnp1jrCMICH/NOVOPlasty/NOVOPlasty/M_R2.fastq


Heteroplasmy:
-----------------------
MAF =
HP exclude list =
PCR-free=


Optional:
-----------------------
Insert size auto= yes
Use Quality Scores= no


Then you will want to create a submission file (.sh file) it should look like this: 


#!/bin/sh --login
#SBATCH --time=5:00:00
#SBATCH --nodes=1
#SBATCH --cpus-per-task=20
#SBATCH --mem=250G
#SBATCH --mail-user=zehnp1jr@cmich.edu
#SBATCH --mail-type=FAIL,BEGIN,END
#SBATCH -J A_Mitochondrial




cd ${SLURM_SUBMIT_DIR}


perl NOVOPlasty4.3.1.pl -c config.txt




scontrol show job $SLURM_JOB_ID


Then you want to submit the job. If using a cluster its a good idea to submit as a job rather than run in the terminal as you will most likely run out of resources and your task will be terminated in the terminal. 
SECTION: Novoplasty v4.3.1
2. Submit consensus file from Artemis to MITOS for Annotation. 

 
This will be the .fasta file from NOVOPLasty. 



SECTION: Novoplasty v4.3.1
3. Next you will want to take annotations from MITOS2 and manually check gene boundaries in Artemis 

 

SECTION: Novoplasty v4.3.1
4.     
Next I followed these steps for my submission to NCBI. 
SECTION: Novoplasty v4.3.1
5. Phylogenetic tree building with protein coding gene data
TITLE: Intradermal coinfection of mice ears with L. major IA-2 and Staphylococcus aureus Newman strain
AUTHORS: Tiffany Borbon, Corey Parlet, Gwendolyn Clay, Mary E. Wilson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Cutaneous coinfection of bacteria with </span><span style = "font-style:italic;">Leishmania</span><span> species in mice provides a model for studying the immune effects of non-sterile inoculation of </span><span style = "font-style:italic;">Leishmania</span><span> protozoa.  In this protocol, we describe a method for intradermal coinfection of mice ear pinnas with </span><span style = "font-style:italic;">Leishmania major </span><span>IA-2 and a low dose of </span><span style = "font-style:italic;">Staphylococcus aureus</span><span> Newman strain. This protocol describes the methods for </span><span style = "font-style:italic;">S. aureus</span><span> and </span><span style = "font-style:italic;">L. major</span><span> preparation, mixing, mouse anesthesia and intradermal injection as well as infection monitoring and euthanasia. This protocol can be modified for the co-inoculation of other bacterial or leishmanial species in mice. </span></div></div>

[STEPS]
TITLE: Dephosphorylation of 5´-ends of DNA using CIP (M0290)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal Alkaline Phosphatase  (CIP - M0290).

[GUIDELINES]
Dephosphorylation of 5' -ends of DNA in Restriction Enzyme Reaction
The phosphate can be added directly into the digestion reaction during or after DNA digestion
CIP is active in all NEB restriction enzyme buffers
DNA purification is required before ligation

[STEPS]
1. Prepare a 20 µL reaction as follows:
   DNA 1 pmol of DNA ends*   CutSmart®  Buffer (10X) 2 μl   CIP 1 unit   H2O, purified  to 20 μl**   

2. Incubate at 37 °C for 30 min.
3. Purify DNA by gel purification, spin-column (NEB #T1020 or NEB #T1030) or phenol extraction.
TITLE: Organoid Immunohistochemistry on Ventana Benchmark XT
AUTHORS: gustavo.parfitt, kristen.whitney

[DESCRIPTION]
Organoid Immunohistochemistry on Ventana Benchmark XT

[STEPS]
SECTION: Immunohistochemistry of organoid sections
1. Fix organoids in 10% NBF for 15-30 minutes.
SECTION: Immunohistochemistry of organoid sections
2. Prepare paraffin-embedded blocks on tissue processer. 
SECTION: Immunohistochemistry of organoid sections
3. Cut 5μm sections from FFPE (formalin-fixed paraffin-embedded) blocks using microtome. 
SECTION: Immunohistochemistry of organoid sections
4. Mount sections on positively charged slides and bake overnight at 70° C. 
SECTION: Immunohistochemistry of organoid sections
5. Prepare primary antibody dilution kit (depending on antibody concentration) for Ventana Benchmark XT automatic staining platform (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. 
SECTION: Immunohistochemistry of organoid sections
6. Load slides onto Ventana and run through alcohol, antigen retrieval, primary antibody incubation and  visualization steps. 
SECTION: Immunohistochemistry of organoid sections
7. Coverslip slides and let dry overnight.  
SECTION: Digital Histopathologic Analysis in QuPath
8. Scan slides at 40x on Aperio VERSA 8 (Leica Biosystems, Wetzlar Germany) digital slide scanner. 
SECTION: Digital Histopathologic Analysis in QuPath
9. Manually segment organoids to generate annotatios in QuPath (version 0.2.3,https://QuPath.github.io/).
SECTION: Digital Histopathologic Analysis in QuPath
10. Perform color deconvolution and determine appropriate settings for positive-pixel-based analysis.
TITLE: Drosophila Locomotion Protocol
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol is used to assay the locomotion of flies as a screening tool. 

[STEPS]
SECTION: Day -10: Set up crosses
1. Set up fly crosses as needed.
SECTION: Day 0-6: Collect progeny
2. ~10 days after setting up the crosses, progeny will begin to eclose. Once daily, collect those that are desired, discarding any undesired genotypes. Flies should be collected in vials with 9-14 flies per vial. 
 
6 replicates per genotype is desirable for the assay, so once 6 vials (of 9-14 flies each) have been collected, the crosses may be discarded even if they are still producing additional flies. 
 
Note: Flies are sensitive to the effects of anesthesia, so collecting them at the time of eclosion is the only time they should be exposed to CO2.  
SECTION: Day 1-10: Aging flies
3. Flies should be flipped (without anesthesia) to new food every 2-3 days while aging and stored at 25 °C in an incubator. 

SECTION: Day 10*: Locomotion assay
4. Supplies needed: 
Timer
Empty vials and buzz plugs
White paper
iPhone or iPod, or alternate recording device, optional 
 
*Day 10 is a standard time point but the assay can be performed at any desired timepoint and will show age related decline in locomotion over at least the course of 3-20 days.  
SECTION: Day 10*: Locomotion assay
5. Perform assay in groups of 4-7 vials at a time when possible. For example, if 30 vials are to be assayed on one day, divide them into 5 groups of 6 or 6 groups of 5.
SECTION: Day 10*: Locomotion assay
6. Flip flies to new polystyrene or polypropylene vials, without food, plugged with a Buzz Plug. Write the genotype or code on the bottom of the vial so that it does not interfere with seeing the flies when the tube is on its side. 
SECTION: Day 10*: Locomotion assay
8. Start Timer. Briefly but firmly tap the first vial 3 times, strong enough to knock the flies to the bottom on the vial, then immediately lay it on its side on top of a white piece of paper and note the time on the timer. Wait 15 seconds, watching the time count down on the timer. 
SECTION: Day 10*: Locomotion assay
7. Allow flies to acclimate for 1 min before beginning.

SECTION: Day 10*: Locomotion assay
9. After the 15 second delay, record a 3 second video of the flies moving in the vial using an iPhone or iPod or equivalent. 
(As an alternative, counts can be performed manually). 
SECTION: Day 10*: Locomotion assay
10. After the video is recorded, put the vial upright and set aside for now. 
SECTION: Day 10*: Locomotion assay
11. Repeat steps 8-10 for each additional vial, until each vial has been recorded 1 time. Then start back again with vial #1, completing a total of 6 sets of recordings. 
SECTION: Day 10*: Locomotion assay
12. At the end of the assay flies are anesthetized and fixed or frozen as needed for downstream assays. 
SECTION: Day 10*: Locomotion assay
13. Analyze movement data in a blinded fashion.
TITLE: iPSC editing with TALENs
AUTHORS: Ruilin Tian, Jason Hong, Martin Kampmann

[STEPS]
?. [Pre-coating 6 well plate]
Pre-coat 6 well plate with Matrigel (diluted 1:50 with Knockout DMEM) by adding  to each well.
1 ml
?. [Passaging and Plating iPSC Cells]
Grow iPSCs until they are ~ 85 % confluent in one well of a 6 well plate or larger format.
?. [Passaging and Plating iPSC Cells]
Remove old medium and wash iPSCs with DPBS 1x.
?. [Passaging and Plating iPSC Cells]
Singularize the cells by gently pipetting them up and down several times.
?. [Passaging and Plating iPSC Cells]
In a 15 ml conical tube, add DPBS and then add the lifted cells in Accutase.
?. [Passaging and Plating iPSC Cells]
Spin down at  for .
Centrifuge: 200 34
?. [Passaging and Plating iPSC Cells]
Aspirate supernatant and resuspend cells in Stemflex.
?. [Passaging and Plating iPSC Cells]
Perform cell count.
?. [Passaging and Plating iPSC Cells]
Remove the Matrigel in the pre-coated plate and add appropriate amount of Stemflex medium with Rock inhibitor (1000x).
?. [Passaging and Plating iPSC Cells]
Re-seed 0.5 M cells into a 6 well plate so that it can reach ~ 60 % confluency the next day.
?. [Passaging and Plating iPSC Cells]
Put the plate in incubator and culture overnight.
?. [Transfection]
The following day, change the media with new E8 media.
?. [Transfection]
Prepare the following mixes:Tube 1: Tube 2:DNA mix ( –  total)        Optimized TALENS ratio            -             -             -             -
[Opti-Mem]
[Lipofectamine Stem Reagent:]
[Opti-Mem]
0.5 µg
5 µg
[Your Plasmid of choice]
[TALENS L]
[TALENS R]
[BCL-XL]
?. [Transfection]
Add tube 2 to tube 1 and mix well.
?. [Transfection]
Incubate the mixture for .
?. [Transfection]
Add the whole mixture to your cells in the 6 well plate.
?. [Transfection]
Incubate overnight and change the media to Stemflex the next day.
?. [Transfection]
If confluent, passage and expand them into 10 cm plate.
?. [Transfection]
When confluent in 10 cm plate, continue to selection protocol.
?. [Pre-coating 6 well plate]
Let it sit in incubator for at least .
?. [Passaging and Plating iPSC Cells]
Add Accutase for  –   to lift the cells.
TITLE: Adverse Events (Part 8 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 8 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: Quantifying Biogenic Silica (bSi) Deposition Rates Adapted Method  & Fluorescence Reading (PDMPO) via Fluorometer
AUTHORS: Brittany Zepernick, Matthew Saxton, Steven Wilhelm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method can be used to assess and quantify the rate of silica deposition (bSi) over time in diatoms to determine their rate of frustule synthesis. This protocol has been adapted for the processing of both cultures as well as environmental samples when inoculated with PDMPO [2‐(4‐pyridyl)‐5‐((4‐(2dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole], which is a fluorescent dye that is co-deposited with silica during frustule biosynthesis in a 3230:1 Si:PDMPO (mol:mol) ratio. </div></div>

[STEPS]