eamag/protocols-with-steps huggingface dataset:
TITLE: Single cell RNA Sequencing for tissue samples processed with microfluidic device platform
AUTHORS: Jeremy Lombardo

[DESCRIPTION]
Protocol for single cell RNA sequencing of tissue samples processed with a microfluidic device platform. 

[STEPS]
1. Euthanize mice (12 week, male, C57BL/6 for kidney; 10 week, female, MMTV-PyMT for mammary tumor, both from Jackson Laboratory, Bar Harbor, ME) by CO2 inhalation.
2. Dissect kidneys or mammary tumor and mince into ~1 mm3  pieces.
3. Process samples using microfluidic device platform.
4. After device processing, centrifuge recovered cells (400xg, 5 min) and treat with 100 Units of DNase I for 5 min at 37°C, and wash by centrifugation into PBS+1% BSA (PBS+).
5. Incubate samples with RBC lysis buffer for 5 min on ice, centrifuge, and resuspend in PBS+.
6. Stain cells with SytoxBlue (Life Technologies, Carlsbad, CA, USA) prior to FACS (FACSAria Fusion, BD Biosciences, Franklin Lakes, NJ) to remove dead cells and ambient RNA.
7. Centrifuge sorted live single cells (SytoxBlue-neg) and resuspend at a concentration of 1000 cells/µL in PBS with 0.04% BSA. ‘
8. Use the 10x Chromium system (10x Genomics, Pleasanton, CA) for droplet-enabled scRNA-seq. Load oil, cells, reagents, and beads onto an eight-channel microfluidic chip. Load lanes with ~17,000 cells from each sample, as determined using an automated cell counter (Countess II, Invitrogen, Carlsbad, CA).
9. Perform library generation for 10x Genomics Single Cell Expression v3 chemistry according to manufacturer’s instructions.
10. Sequence the samples at the desired depth using an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA).
11. Align sequencing fastq files using 10x Genomics Cell Ranger software (version 3.1.0) to an indexed mm10 reference genome.
12. Use Cell Ranger Aggr to normalize the mapped reads for cells across the libraries for each data set.
13. Discard genes that were not detected in at least 3 cells from further analysis. Discard cells with low (3000 for kidney; >4000 for mammary tumor) unique genes expressed, as these potentially represent low quality or doublet cells, respectively. Discard cells with high mitochondrial gene percentages (>50% for kidney and >25% for mammary tumor), as these can also represent low quality or dying cells.
14. Use the Seurat pipeline for cluster identification. Perform principle component analysis (PCA) using genes that are highly variable. Perform density clustering to identify groups and use Uniform Manifold Approximation and Projection (UMAP) plots to visualize the groupings.
TITLE: 5 Methods for DNA-protein imaging by AFM in fluid
AUTHORS: Philip J. Haynes, Kavit H. S. Main, Alice Pyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 5 of the "Atomic Force Microscopy of DNA and DNA-Protein Interactions" collection of protocols.</div><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and it’s interactions with proteins.</span></div></div>

[STEPS]
?. [Methods for DNA-protein imaging by AFM in fluid]
Prepare a mica substrate as described in protocol 1.
?. [Methods for DNA-protein imaging by AFM in fluid]
PEGylate the substrate and adsorb DNA/DNA-protein as described in protocol 2, method 2.3. Place the sample in the AFM.
?. [Methods for DNA-protein imaging by AFM in fluid]
Setup the AFM and approach the DNA sample surface as described in protocol 3.
?. [Methods for DNA-protein imaging by AFM in fluid]
Optimise imaging parameters (protocol 4) such that the DNA and DNA-protein complexes are sufficiently tracked.
?. [Methods for DNA-protein imaging by AFM in fluid]
If inadequate DNA tracking persists due to the influence of the brush-like PEG chains, increase the PeakForce Setpoint (see Note 27) and PeakForce Amplitude. Adjust and optimise the Lift Height accordingly (see Note 26).
?. [Methods for DNA-protein imaging by AFM in fluid]
All other imaging parameters are synonymous to those stated in protocol 3 and protocol 4 when approaching and optimising. A non-exhaustive list of parameters when using an Fast-Scan D are outlined in Table 1.  AB1PFT ParametersTypical value2Scan Size [nm]120 - 2503Pixel density [pixels/line]256 - 5124Line rate [Hz]3-55Imaging PeakForce Setpoint [pN]~ 70 *6PF acquisition Frequency, see Note 19 [kHz]87LP Deflection Bandwidth [kHz]208Sync Distance, see Notes 19 and 21 [μs]70 / 209PeakForce Amplitude, Note 22 [nm]5-10 *10Lift Height, see Note 26 [nm]5-711Z Range (Z Limit if using MultiMode® 8) [nm]500-100012Deflection Limit [V]⋜12.24
AB1PFT ParametersTypical value2Scan Size [nm]120 - 2503Pixel density [pixels/line]256 - 5124Line rate [Hz]3-55Imaging PeakForce Setpoint [pN]~ 70 *6PF acquisition Frequency, see Note 19 [kHz]87LP Deflection Bandwidth [kHz]208Sync Distance, see Notes 19 and 21 [μs]70 / 209PeakForce Amplitude, Note 22 [nm]5-10 *10Lift Height, see Note 26 [nm]5-711Z Range (Z Limit if using MultiMode® 8) [nm]500-100012Deflection Limit [V]⋜12.24
TITLE: Making LB NGM Plates
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. Sometimes they can also be grown on LB-NGM, where the LB in the media will promote better growth of the bacteria the worms feed on. </div><div class = "text-block">The LB-NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of LB-NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another.</div></div>

[STEPS]
?. [Pre-Autoclave:]
Book the autoclave (notebook on top of the machine).
?. [Pre-Autoclave:]
Take clean flasks from the glass kitchen (Only the ones with autoclave tape on are sterile)
?. [Pre-Autoclave:]
Measure all the pre-autoclave reagents and add to the flask (Use a new weighing boat and spatula for each reagent. Also, the cholesterol is kept in the fridge.)
?. [Pre-Autoclave:]
Once water is added mix thoroughly and label with autoclave tape (‘LB NGM Rm 5020’). Makesure the bottle is not screwed completely when placing it inside the autoclave machine.
?. [Using the Autoclave:]
Turn ON the autoclave
?. [Using the Autoclave:]
Make sure that the autoclave’s probe bottle is the same size as the largest bottle you use and fill it with water.
?. [Using the Autoclave:]
Place the temperature probe in it.
?. [Using the Autoclave:]
Fill up the autoclave with water until it reaches the grill.
?. [Using the Autoclave:]
Place the bottles in the autoclave and make sure that the cap is not screwed completely.
?. [Using the Autoclave:]
Check the waste flask is not too full
?. [Using the Autoclave:]
Use ‘media’ program.
?. [Using the Autoclave:]
Press START.
?. [Using the Autoclave:]
It will take about 2.5 hours for 1L or larger bottles.
?. [Post-Autoclave:]
When autoclave is complete, remove the probe flask
?. [Post-Autoclave:]
Make sure to wear gloves as the flask will be hot
?. [Post-Autoclave:]
Let the agar to cool to around 55°C, ie the bottle is cool enough to hold for a second with a gloved hand.
?. [Post-Autoclave:]
Add the post autoclave reagents.
?. [Post-Autoclave:]
Mix it well and start pouring onto desired sized plates (See Protocol for plate pouring)
?. [Post-Autoclave:]
Try not to shake the bottle too much while mixing to avoid air bubbles.
?. [Post-Autoclave:]
The agar needs to be warm to be poured without blocking the tubings, so try to pour as quickly as possible and if not poured immediately put the bottle on a waterbath set to 60C until being used.
TITLE: Soil sample DNA extraction (SuperSoil)
AUTHORS: Hsin-Mao Wu, Jie Hao Ou, Yin-Tse Huang, Yu-Hsuan Fan

[DESCRIPTION]
A method for fungal and bacterial DNA extraction.

[STEPS]
SECTION: Sample preparation &amp; Cell lysis
1. Add 0.2 mL  ⌀=1.0 mm zirconia beads, 0.2 mL  ⌀=0.5 mm zirconia beads, 800 µL  SD1 solution, 0.5 g    into a 1.5 ml microcentrifuge tube. Vortex briefly to mix.
 


 

 

SECTION: Sample preparation &amp; Cell lysis
2. Homogenize samples using Vortex-Genie® 2 with SI-H524 Horizontal microtube holder. Vortex at maximum speed for 4 min 


 

SECTION: Sample preparation &amp; Cell lysis
3. Centrifuge the 1.5 ml microcentrifuge tube at14000 rpm, 2 min, 25 °C 



SECTION: Inhibitor precipitation
4. Add 200 µL  SD2 solution to a clean 1.5 ml microcentrifuge tube.

SECTION: Inhibitor precipitation
5. Transfer 500 µL  supernatant to the tube in the previous step. Vortex 5 s  for mixing.

 

 

SECTION: Inhibitor precipitation
6. Centrifuge at14000 rpm, 2 min, 25 °C 
SECTION: DNA binding
7. Add 600 µL  SD3 solution to a clean 1.5 ml microcentrifuge tube. Avoiding the pellet, transfer up to 700 µL  of supernatant from the previous step to that tube. Vortex 5 s  for mixing.
 


SECTION: DNA binding
8. Take 650 µL  to spin column (with collection tube) and centrifuge at14000 rpm, 2 min, 25 °C  

SECTION: DNA binding
9. Discard the flow-through and repeat step 8 to ensure all the lysates have passed through the spin column.
SECTION: DNA binding
10. Carefully place the spin column into a clean 2 ml collection tube. Avoid splashing any flow-through onto the spin column.
SECTION: Wash
11. Add 500 µL  of Solution SEA to the spin column. Centrifuge at 14000 rpm, 2 min, 25 °C 
 

SECTION: Wash
12. Discard the flow-through and return the spin column to the same 2 ml collection tube.
SECTION: Wash
13. Add 500 µL  80% ethanol to the spin column. Centrifuge at 14000 rpm, 2 min, 25 °C 
 


SECTION: Wash
14. Discard the flow-through and place the spin column into a new 2 ml collection tube.
SECTION: Wash
15. Centrifuge at 14000 rpm, 3 min, 25 °C  Carefully place the spin column into the new 1.5 ml microcentrifuge tube
 


SECTION: Elute
16. Add 50 µL  RNase-free water (DEPC-treated water) to the center of the white filter membrane.
 


SECTION: Elute
17. Centrifuge at 14000 rpm, 2 min, 25 °C  Discard the spin column. The DNA will be in the microcentrifuge tube.

SECTION: Quality and quantity assessment
18. Use BioTek Take3 & Take3 Trio Microvolume Plates to measure the quality and quantity of your specimens. And then upload them to Google Drive (1 MolecularWork (Direct edit) 分生實驗號碼 (直接編輯) w_ Primer list)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Mapping metagenomic reads from <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Da</a>y (OSD) 2014 against NCBI's ViralRefSeq alongside viral sequences identified from the Tara Oceans survey using <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533152/" target="_blank">VirSorter</a>.

[BEFORE_START]
To run this protocol, users must first <a href="https://user.iplantcollaborative.org/register/" target="_blank">register</a> for Cyverse account. All data (both inputs and outputs) are available within Cyverse's data store at /iplant/home/shared/iVirus/ExampleData/

[GUIDELINES]
One of the most commonly used procedures for analyzing viral metagenomic data is to map their reads (or reads from another dataset) against a set of references, often those from the read assembly. For example, if one wanted to know how well-represented viruses in NCBI's Viral Reference Sequences (ViralRefSeq) were in ocean viromes, they could map reads from lots of ocean viral metagenomes against ViralRefSeq. This is generally done using <a href="http://bowtie-bio.sourceforge.net/bowtie2/index.shtml" target="_blank">Bowtie2</a> or <a href="http://bio-bwa.sourceforge.net/" target="_blank">BWA</a>, by selecting a reference set of sequences, and then providing paired or unpaired reads to Bowtie2/BWA. <em>Then</em> the results must be processed/filtered to generate coverage tables. Dealing with setting up multiple reads files (10 paired metagenomes = 10 alignment runs) and the processing those read files can be challenging (not to mention requring computational resources).<br /><br />In this protocol, we'll be using reads fromÂ <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Da</a>y (OSD) 2014 and map them against ViralRefSeq alongside viral sequences identified from the Tara Oceans survey using <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533152/" target="_blank">VirSorter</a>.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Quantification of p62-positive inclusions
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Quantification of p62-positive inclusions in the rodent brain

[STEPS]
1. Scan the 5um sections immunostained with guineapig anti-p62 (1:500, Progen #GP62-C), mouse anti-α-synuclein (1:500, BD Biosciences #610786) and TH (1:1000, Calbiochem #657012) using ZEISS LSM 980 with Airyscan 2. 
2. Visualize the images with ZEN 2011 software (Zeiss, Germany) and determine one representative coronal section of the brain region of interest.
3. Determine the total number of p62-immunopositive Marinesco bodies (nuclear inclusions)
and Lewy body-like inclusions (cytoplasmic inclusions) falling into each category (Syn-positive or Syn-negative) in that representative section.
TITLE: Cross-cultural adaptation and validity of the Italian version of the Anterior Knee Pain Scale: a prospective multi-centre study
AUTHORS: Jacopo E. Rocchi, Pasquale Alessio Sauchelli, Sebastiano Nutarelli, Lorenzo Rum, Riccardo Ciatti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Purpose: validating and culturally adapting the Anterior Knee Pain Scale (AKPS) into Italian.
</div><div class = "text-block">The AKPS is used for the clinical evaluation of patients with anterior knee pain. By using stricter methodological criteria, this study aims at reviewing the first validation attempt made by Cerciello et al., 2018.
The manuscript will be developed following the COSMIN checklist.</div><div class = "text-block">The optimization of the cultural adaptation and survey translation will be achieved through the independent translation of a healthcare professional and a member of the general public. Both translators hold an official English language certificate, level C1 or higher.
Possible difference between the translations will be resolved through discussion with a reunion between translators and authors (health professionals and an orthopedic surgeon with particular interest in knee rehabilitation).</div><div class = "text-block">This draft will be then sent to an English-speaking translator for a back translation.
The back-translation was performed by an italian-english professional translator blinded to the original document.
The committee, composed of both the authors and the translators, will then meet to finalize the final version of the AKPS.</div><div class = "text-block">The study protocol was approved by the Institutional review board of the University of Rome “Foro Italico”.
Patients aged between 18 and 60 years with patellofemoral pain syndrome, patellar dislocation or lateral subluxation will be recruited and evaluated through self-reported questionnaires.
Individuals with cognitive impairments and concomitant injuries to the affected limb, including anterior or posterior cruciate ligament injury and meniscal injury will be excluded.</div><div class = "text-block">Following the guidelines, the minimum sampling will be of 130 patients, or 10 subjects multiplied by the number of items in the questionnaire. 
Before proceeding, a pilot study with a preliminary analysis of the data will be carried out with a sample of 25 subjects.</div><div class = "text-block">Data will be collected in digital format, extracted, and processed in a computerized manner.
Cronbach's alpha for internal consistency and intraclass correlation coefficient (ICC) for test-retest reliability will be used as statistical analysis to assess the reliability of the translation.</div><div class = "text-block">Concurrent and construct validity will be evaluated by comparing the AKPS results with well-established, Italian-validated scales. 
The above-mentioned scales will be the Knee Injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee Subjective Knee Form (IKDC), the SF-36 Health Survey, the Victorian Institute of Sport Assessment–Patella (VISA-P), and the Visual Analog Scale (VAS).</div></div>

[STEPS]
TITLE: Single-cell suspension preparation from Human bronchial biopsies to perform scRNA-sequencing using 10x chromium
AUTHORS: Martijn Nawijn, Leonie Apperloo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol enables the dissociation of human bronchial biopsies into a single-cell suspension starting from fresh materials. These single cells are suitable for use in 10x chromium kits for single-cell RNA sequencing.</div></div>

[STEPS]
?. [Before you start]
All steps should be performed in a sterile environment. Prepare all the buffers and reagents at room temperature as described in the "Materials" section.
?. [Before you start]
Freshly prepare the tissue dissociation solution.Dilute the 10x  RBCs lysis buffer to 1x.
?. [Preparation of single-cell suspension]
Directly proceed by transferring the biopsies to a petri dish using sterile tweezers.
?. [Preparation of single-cell suspension]
Cut the biopsies using a scalpel into smaller pieces at room temperature.
?. [Preparation of single-cell suspension]
Transfer the pieces to the FACS tube containing 1ml of tissue dissociation solution (HBSS +1%P/S + Collagenase D + DNaseI). Rinse the petri dish with tissue dissociation solution to collect all the materials.
?. [Preparation of single-cell suspension]
Incubate for 1 hour @37°C, shake regularly. After this incubation keep the samples on ice.
?. [Preparation of single-cell suspension]
Strain the mixture through a 70μm filter and collect the filtrate in a 50ml tube while keeping the samples on ice. Push the sample through the filter using the back of a plunger. Rinse the FACS tube with 1 ml cold HBSS+1%P/S and add it to the filter. Wash the filter with 1 ml HBSS+1%P/S. Collect any remaining cell suspension materials from the back of the filter using a pipette.
?. [Preparation of single-cell suspension]
Centrifuge @590xg, 5 min at 4°C .
?. [Preparation of single-cell suspension]
Resuspend the pellet in 500 μL resuspension buffer (PBS+0,04% BSA) and transfer the sample to a 1.5ml Low-Bind tube. Add 1 ml RBCs lysis buffer (1x) and incubate for ~2-5 minutes on ice.
?. [Preparation of single-cell suspension]
Centrifuge @590xg, 5 min at 4°C.
?. [Preparation of single-cell suspension]
Resuspend the pellet in 50μL resuspension buffer (PBS+0,04% BSA).
?. [Preparation of single-cell suspension]
Count the cells using Trypan blue staining (1:1 ratio of Trypan blue to sample) and a manual hemocytometer. While keeping the rest of the cells on ice.
?. [10x single-cell RNA-seq kit instructions]
Continue with 10x Chromium (Next GEM Single Cell 3ʹ reagent kit) and load 10,000 cells on a single lane.
?. [Before you start]
Collect the tissue in a coulter counter cup filled with 15ml cold PBS (1x). Keep the tissues on ice before starting.
?. [Preparation of single-cell suspension]
Proceed with 10x chromium protocol if 80-90% of the cells are alive.
TITLE: Characterization
AUTHORS: Hung Liang Pai

[STEPS]
?. [In vitro protein synthesis]
Add the reagents(on ice) for synthesis of mRFP into one well of the 384-well plate. Add the reagents from top to down.   ABCD1OrderLocationReagentAmount21Upper-leftDNase/RNase free waterTill 30 μL32Upper-leftSolution A12μL43Lower-rightSolution B9μL54Lower-rightRNase inhibitor1.2μL65Lower-leftRFP+terminator300ng7total30μLDo triple repeat
ABCD1OrderLocationReagentAmount21Upper-leftDNase/RNase free waterTill 30 μL32Upper-leftSolution A12μL43Lower-rightSolution B9μL54Lower-rightRNase inhibitor1.2μL65Lower-leftRFP+terminator300ng7total30μL
Pipetting Solution A, Solution B, RNase inhibitor before adding.Pipetting the eppendorf contain RFP+terminator template to prevent precipitation.
?. [In vitro protein synthesis]
Seal the plate with microseal (on ice).
?. [In vitro protein synthesis]
Centrifuge
Centrifuge: 4000 33, 1 min, 4 10
?. [Setup plate reader 1]
Plate type: 384 well plateSelect wells: At run timeDescription: 1.Temperature
37 °C
?. [Incubation]
Put the plate into plate reader.Incubate the mixture and measure the fluorescence in plate reader follow Setup 1 for
?. [Addition of Buffer]
Take the 384 well plate out of the plate reader.
?. [Addition of Buffer]
Apportion 5 μL of mRFP reagent into 18 well.
Pipetting before apportion.We actually add 4 μL into every well since we think it may evaporate.
?. [Addition of Buffer]
Add pH2,5,7,8,10,12 Buffer solution respectively into different wells of mRFP till total volume of 100 μL.Do triple repeat.
Pipetting after adding buffer solution
?. [Setup Plate Reader 2]
Plate type: 384 well plate
?. [Setup Plate Reader 2]
Temperature:
37 °C
?. [Setup Plate Reader 2]
Excitation start: 550nm, Stop: 620 nmFixed emission: 650 nmStep: by 1 nmGain: Auto Gain (measured with selected wells)
?. [Setup Plate Reader 2]
Emission start: 550nm, Stop: 700 nmFixed excitation: 540nmStep: by 1 nmGain: Auto Gain (measured with selected wells)
?. [Measuring]
Measure the fluorescence excitation and emission intensity of mRFP
?. [Addition of Buffer]
Centrifuge
Centrifuge: 4000 33, 1 min, 4 10
?. [Addition of Buffer]
Centrifuge
Centrifuge: 4000 33, 1 min, 4 10
?. [Addition of Buffer]
Seal the well.
?. [Addition of Buffer]
Remove its seal.
TITLE: Immunocytochemistry
AUTHORS: anita.adami

[DESCRIPTION]
This protocol describes how to perform immunocytochemistry on 2D fixed cells 

[STEPS]
SECTION: Fixing
1. The cells were washed three times with DPBS (GIBCO) and fixed for 10 minutes with 4% paraformaldehyde (Merck Millipore), followed by three more rinses with DPBS.
SECTION: Blocking and permeabilisation
2. The cells were blocked for 60 min in blocking solution (KPBS 0.25% Triton X-100 (Fisher Scientific) and 5% normal donkey serum) at Room temperature.
SECTION: Staining
3. The primary antibody was added to the blocking solution and incubated 60 min at Room temperature (follow manufacturer's instructions for dilutions).
SECTION: Staining
4. The cells were then washed the next day three times with KPBS 0.25%.
SECTION: Staining
5. The secondary antibody was added with DAPI (1:1000; Sigma-Aldrich) to the blocking solution and incubated at Room temperature for 60 min.

SECTION: Staining
6. The cells were finally rinsed 2-3 times with KPBS and visualised on a Leica microscope (model DMI6000B).
TITLE: Inductively coupled plasma mass spectrometry (ICP-MS)
AUTHORS: An.Huang

[DESCRIPTION]
Inductively coupled plasma mass spectrometry (ICP-MS) can be used to detect metal elements existing in samples. Because we are not capable of conducting ICP-MS on our own, we must seek help from our advisors. This protocol is a general description of our actions.

[STEPS]
1. Filter all samples to be tested using 0.22μm syringe filters.
 


2. (Optional) Dilute the samples to 0-20ppb using nitric acid (2%) if it contains too much desired elements more than the measuring range of equipment.
3. Asking an assistant for help in conducting ICP-MS. Obtain a standardized curve before starting to measure the samples.