eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Aims: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome - decreased ventricular function. Titin (TTN) - a giant protein, expressed in cardiac and skeletal muscles, is an important part a fundamental part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS). Methods and Results: iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to -adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells. Conclusions: These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

[STEPS]
SECTION: Generation of patient-specific induced pluripotent stem cells
?. 
SECTION: Karyotype analysis
?. 
SECTION: Teratoma formation
?. 
SECTION: Genotyping
?. 
SECTION: Immunofluorescence staining
?. 
SECTION: Action potential recordings
?. 
SECTION: Extracellular electrograms recorded from spontaneously contracting EBs and analysis of Beat Rate Variability (BRV)
?. 
SECTION: Measurements of intracellular Ca2+ transients and contractions
?. 
SECTION: Mass Spectrometry sample preparation and data processing
?. 
SECTION: Transmission electron microscopy
?. 
SECTION: Statistical analysis
?. 
SECTION: Immunocytological analysis in iPSC-CM
?.
TITLE: DNA extraction - Zooplankton - 96 wells
AUTHORS: coline.royaux, Nicolas Rabet, Céline Bonillo

[DESCRIPTION]
This protocol was used to extract DNA from whole or parts of zooplanktonic freshwater crustaceans (Copepoda, Branchiopoda, ...) from New Caledonia.

[STEPS]
1. Prepare your 96-well extraction plate with one individual per well. Alternate genus in the wells to detect eventual contamination between wells.
1.1. Collect one individual from a sample
1.2. Note its genus and determine its sex with a binocular microscope
1.3. For big individuals (more than 5 mm), dissect a few legs and put it in the well. Be careful not to damage the rest of the body and put it in a tagged Eppendorf tube.
For little individuals (less than 5 mm), put the whole body.
 

1.4. When all 96 wells are filled, the biological material has to dry to go to lysis

 

 



2. Prepare the lysis
2.1. Mix 18 mL and 2.5 mL in a Multi-Channel Reservoir and distribute 200 µL with a multimicropipette in each well
 

2.2. Close your extraction plate with a heated aluminium foil and an adhesive plastic film
3. Put your plate in a proofer at 56 °C    to lyse the tissues
4. Perform the DNA extraction with a DNA extraction robot
4.1. Remove the adhesive film and aluminium foil from the plate and put it in the robot
4.2. It will deposit 200 µL and 200 µL in each wells and mix it

4.3. Then, 600 µL are transfered on the tissue binding plate. Reagents excess are emptied in a waste container.

4.4. The tissue binding plate is then dried by a 5 min aspiration to bind DNA to the silica membrane of the binding plate
4.5. The silica membrane is then washed with 600 µL and twice with 900 µL per well. Each wash is intercalated by a 5 min aspiration dry.

25. The waste container is removed from under the binding plate which is dried again by a 10 min aspiration.

25. An empty extraction plate is placed under the binding plate to retrieve the genomic DNA from the binding plate
25. 
4.6. The waste container is then removed from under the binding plate which is dried again by a 10 min aspriration

4.7. An empty extraction plate is placed under the binding plate to retrieve the genomic DNA from it
4.8. DNA is eluted from the tissue binding plate with 100 µL in each well and is collected in the new extraction plate underneath

4.9. After a 3 min rest, the binding plate is dried for 2 min and the elution is repeated with 100 µL 

4.10. Retrieve the new extraction plate containing the genomic DNA and discard the rest
TITLE: DNT Detection In Soil
AUTHORS: Zhujun Wei

[STEPS]
?. The 1% bacteria (PYB1s-yqjF3rd-luxCDABE) were cultured in LB containing 0.1% streptomycin (50 mg/mL) for 24 h with aeration at 37°C to reach a final density of 1 × 108 cells/mL.
?. Preparation of 2% Alginate solution, 1% calcium-chloride solution, experimental soil:dissolve 2 g Alginate in 100 mL distilled water (2% w/w) and 1g calciumchloride in 100 mL distilled water (1% w/w). The experiment soil was filtrated prior to use.
?. Sterilize the 2% Alginate solution, 1% calcium-chloride solution, (121°C, 15min), and experimental soil (121°C, 1.5h).
?. Add 0, 0.25, 0.5 and 1 mg of DNT powder (0, 250, 500 and 1000 mg/kg) to the wells of a 12-well plate.Take 1g of experimental soil and drop it onto the DNT in the 12-well plate on a clean bench.
?. The engineered bacteria were added into 2% Alginate solution at a 1:9 (v/v) ratio.
?. Drop 200 µL of 1% (w/w) calcium-chloride solution into the 1 mL alginate-bacterial suspension droplets (the volume ratio between the Alginate mixture and calcium chloride solution was 5:1). Leave the mix for 1 minute.
?. The mixed Alginate gel was tiled on the soil.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes a clean up and size selection method for nucleic acids (tested on DNA) to deplete and remove fragments below 1 - 2 kb. <br />The success of this depends on the cleanliness of your sample (it doesn't have to be super clean but a whole lot of contaminants make working with the beads more difficult, diluting the sample out before usage can help with that). </p>
<p> </p>
<p>The concentrations of PEG and NaCl and the volume of the beads solution are crutial for recovery and proper removal of small fragments. </p>
<p>As a basic guidline it can be said: more PEG and NaCl - higher recovery but hence less removal of small fragments and the other way round. I found for my samples (eucalyptus) that with 1 volume of the beads solution respectively to DNA sample I'm on the safe side recovery wise, but if I want to make sure to get rid of more smaller fragments I use 0.8 volumes.</p>
<p> </p>
<p>So in numbers that means: </p>
<p>Final reaction concentration of PEG8000: </p>
<p>1 V:    5.5%    <br />0.8 V: 4.8%</p>
<p> </p>
<p>Final reaction concentration of NaCl: </p>
<p>1V: 0.8 M</p>
<p>0.8V: 0.7 M</p>
<p> </p>
<p> </p>

[BEFORE_START]
<p>Before doing this clean up on your whole (maybe very precious) sample I would recommend testing it out on a smaller amount (300 - 500ng) to make sure the beads work for you. </p>
<p> </p>
<p>- For making the beads solution stock take Sera-Mag SpeedBeads (<a href="http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-au/products/AlternativeProductStructure_24411/29103105" target="_blank">GE Healthcare</a>, PN 24152105050250 ) out of the fridge and let warm up to room temperature </p>
<p> </p>
<p>- For further clean ups always take the beads stock solution out of the fridge at least 15 minutes before usage to let it warm up to room temperature  and mix (vortex) very well until soluion looks homogeneous and no bead clumps are visible anymore (that is very important) </p>
<p> </p>
<p>- Always make fresh 70 % Ethanol</p>
<p> </p>
<p>- Preheat your elution buffer of choice (TE-Buffer, Tris 10 mM, Water..) to 50<strong>°</strong> until usage </p>

[GUIDELINES]
<p> </p>
<p>Beads solution adapted from Philippe Jolivet and Joseph W. Foley, 2015 - Solutions for purifying nucleic acides by solid-phase reversible immobilization (SPRI))</p>
<p>Link: http://www.openwetware.org/images/f/f8/SPRI_buffers_v2_2.pdf </p>
<p>There really accurate and helpfull recipies for making up every ingredient of the beads solution can be found, I just adapted the concentrations for my needs. </p>
<p> </p>
<p>Thank you Benjamin Schwessinger for supporting and encouraging me to try every single concentration combination out there to find a simple and well working one. </p>
<p> </p>
<p>I also use the same beads solution for the MinION sequencing library preparation. </p>

[STEPS]
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TITLE: Modified nuclease flush protocol for Nanopore RNA flow cells 
AUTHORS: Anthea Hull, Ricardo De Paoli-Iseppi, Mike Clark

[DESCRIPTION]
A nuclease flush can be used to recover blocked pores on Oxford Nanopore Technologies (ONT) flow cells to maximise overall sequencing output. The Flow Cell Wash Kit (EXP-WSH004/EXP-WSH004-XL) was developed to remove blockages on DNA flow cells, but does not restore blocked pores on their RNA counterparts. This short protocol describes a simple modification to the Nanopore Flow Cell Flush protocol to enable pore recovery in RNA flow cells. 


 

This protocol uses two RNases to digest blockage-causing nucleic acids: RNase CocktailTM Enzyme Mix and RNase H. RNase CocktailTM contains endoribonucleases RNase A and RNase T1, which cleave at C and U, and G residues, respectively. RNase H, rather than cleaving single-stranded RNA, degrades the RNA strand of DNA-RNA hybrid molecules, allowing it to target the hybrid libraries used for direct RNA sequencing. When used in combination with WMX (a DNase I containing solution provided in the ONT EXP-WSH004 wash kit), blockage-causing DNA, single-stranded RNA or DNA-RNA hybrids can be degraded. 

 

Based on our preliminary results, this protocol can recover up to 50% of available pores present at the start of a run, and can boost the number of sequenced bases by >50%. Additionally, flushing RNA flow cells in accordance with this protocol has a negligible effect on read quality or length.
 


[STEPS]
SECTION: Protocol
1. Thaw Wash Mix (WMX), RNase H and RNase CocktailTM on ice. Do not vortex.
SECTION: Protocol
2. Thaw one tube of Wash Diluent (DIL) at room temperature.
SECTION: Protocol
3. Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
SECTION: Protocol
4. In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:
 

 

SECTION: Protocol
5. Mix well by pipetting, and place on ice. Do not vortex the tube.
SECTION: Protocol
6. Follow Oxford Nanopore EXP-WSH004/EXP-WSH004-XL Protocol (accessible from link below) from Step 6 as directed, using the prepared Flow Cell Wash Mix to complete the flow cell flush.
 
EXP-WSH004/EXP-WSH004-XL Protocol: 
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/flow-cell-wash-kit-exp-wsh004/v/wfc_9120_v1_revq_08dec2020/flushing-a-promethion-flow-cell?devices=promethion
SECTION: Protocol
7. Optional: Following the 1 hour incubation and waste removal steps as described in the standard protocol, we recommend the flow cell be loaded with storage buffer as detailed in the "To store the Flow Cell for later use" protocol and a flow cell check be performed to quantify extent of pore recovery.  

Flow Cell storage Protocol: 
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/flow-cell-wash-kit-exp-wsh004/v/wfc_9120_v1_revq_08dec2020/to-store-the-promethion-flow-cell-for-later-use?devices=promethion
SECTION: Protocol
8. The flow cell may now be stored for later use or used immediately with a fresh RNA library.
TITLE: Cardiac Action Potential Protocol
AUTHORS: Robert  Harvey, Shailesh Agarwal

[DESCRIPTION]
Cardiac Action Potential Protocol

[STEPS]
1. Membrane potentials were recorded from isolated pig ventricular myocytes using the whole-cell configuration of the patch clamp technique.
2. Isolated cells were placed in a perfusion chamber on the stage of an inverted microscope and bathed in an extracellular solution containing (in mM) NaCl 140, KCl 5.4, CaCl22.5, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH 7.4), maintained at 37oC. 
3. Cells were patched using microelectrodes with resistances between 1 and 2 MΩ. 
4. Cells were dialyzed with an intracellular solution containing (in mM): K-aspartate 110, KCl 25, NaCl 5, MgATP 3, cAMP 0.002, phosphocreatine dipotassium 10, EGTA 0.01, and HEPES 10 (pH 7.2).
5. Membrane potential was recorded under current clamp conditions using a Multiclamp 700B voltage clamp amplifier, Digidata 1440A computer interface, and pClamp 11 data acquisition and analysis software (Molecular Devices). Data were lowpass filtered at 4 kHz, and sampled at 10 kHz.
6. Action potentials were elicited by applying a depolarizing stimulus (2 ms) once every 5 s. The amplitude of the stimulus was adjusted to approx. 1.2 times the minimum value needed to generate an action potential. 
7. A stable baseline (about 5 minutes) was obtained before application of test drugs.
8. Cells were not used if the resting membrane potential was less than -75 mV, the stimulus artifact overlapped the upstroke of the action potential, or a stable baseline could not be established,
TITLE: DToL Taxon-specific Standard Operating Procedures for the Plant Working Group 
AUTHORS: Laura L Forrest, Michelle Hart

[DESCRIPTION]
This Standard Operating Procedure (SOP) collection contains guidance on how to process the various plant taxa within the scope of the Darwin Tree of Life (DToL) project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Royal Botanic Garden Edinburgh (RBGE)) and outlines the flash frozen plant tissues required for whole genome sequencing (WGS), which takes place at the Wellcome Sanger Institute . Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger Institute.

[GUIDELINES]
Use: These guides outline how to collect and prepare specimens for DNA barcoding, genome sequencing, and specimen vouchering when relevant. Different taxa may require different sampling, preparation, photography, storage techniques and processes. 

Please see the Sanger Sample Submission SOP (link below) for general details on physical handling of specimens, including cold chain use, and collation of metadata for sample submission to Sanger.

Future plans for these SOPs: These SOPs will be reviewed on an ad hoc basis by the Plant Working Group to incorporate feedback from the community.

Raising issues: We are still developing best practice and elements of these SOPs are subject to change. If you would like to share any advice, comments, techniques or lessons learned, please add comments to the protocol or email edtolnumbers@rbge.org.uk.  

Links to SOPs relating to this document:

Sanger Sample Submission SOP v2.2
Sanger Sample Metadata Recording SOP v2.0


Document History [add below]
 
     Version      Date      Major changes      Contributors        1.0      2020-04-07      Draft   version      Lyndall   Pereira (NHM)        2.0      2020-04-29      DNA   barcoding for reference library at NHM edited      NHM: Olga   Sivell, Laura Sivess, Gavin Broad, Ben Price, Lyndall Pereira        3.0      2020-05-13      Addition of   pictorial dissection for each taxon group      Liam   Crowley (Oxford WW) and Lyndall Pereira (NHM)        3.1      2020-07-17       SOP text   split into Order-specific SOPs. Uniform style, publication quality images   with colour blind friendly dissection lines      Olga   Sivell, Laura Sivess        4.0      2022-01-04      Overall   formatting further improved (including spelling/ punctuation changes, table   of contents links), photo guides made more uniform, edits in protocol made to   reflect any adaptations in the sampling process since July 2020, additional   taxon protocols included, unrequired taxon guides removed. First submission   to protocols.io.      Inez   Januszczak (NHM), Liam Crowley (Oxford WW), Olga Sivell (NHM), Laura Sivess   (NHM), Chris Fletcher (NHM)   
 

Acknowledgements: RBGE DToL staff have been involved in looking over and revising these SOPs. 


[STEPS]
TITLE: RNA isolation and qRT-PCR
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the RNA isolation from cultured cells and quantitative RT PCR.

[STEPS]
SECTION: RNA isolation and qRT-PCR
1. Aspirate media from cells and rinse cells with PBS on ice.

SECTION: RNA isolation and qRT-PCR
2. Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.
SECTION: RNA isolation and qRT-PCR
3. Generate cDNA from 1 µg purified RNA using iScript cDNA synthesis Kit (Bio-Rad) according to manufacturer’s protocol.

SECTION: RNA isolation and qRT-PCR
4. The iScript cDNA was diluted 1:10 by using sterile water.
SECTION: RNA isolation and qRT-PCR
5. Combine 10 µL SYBR Green Master Mix (BioRad) with 6.78 µL Sterile Water per sample.

SECTION: RNA isolation and qRT-PCR
6. Combine 16.78 µL diluted SYBR Green Master Mix with 0.61 µL each of 10 micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate 2 technical replicates were ran for each sample.

SECTION: RNA isolation and qRT-PCR
7. Pipette 2 µL of diluted RNA from step 4 in well with SYBR Green Master Mix.

SECTION: RNA isolation and qRT-PCR
8. Cover plate with Optical Adhesive Covers (Applied Biosystems).
SECTION: RNA isolation and qRT-PCR
9. Spin down plate in tabletop centrifuge.
SECTION: RNA isolation and qRT-PCR
10. Run qPCR in CFX96 Real-Time System (BioRad) using the following PCR steps.
SECTION: RNA isolation and qRT-PCR
11. PCR cycle.
 

SECTION: Analysis of qRT PCR data
12. Calculate the ΔCT values by subtracting respective gene CT value from housekeeping gene GAPDH value.
SECTION: Analysis of qRT PCR data
13. Followed by calculating 2ΔCT values, normalize the mRNA expression levels for genes of interest to GAPDH and represented relative to control.
TITLE: IMARIS Analyses of LAMP_CATD in vitro
AUTHORS: Chiara Pavan, Stefano Frausin, Clare Parish, Lachlan Thompson

[DESCRIPTION]
This protocol details how to analyse in-vitro LAMP and cathepsin D staining using Imaris software.

[BEFORE_START]
Select your image files folders. Drag the .LIF files into the Imaris Arena.
Double click. Do not open \LNG files

[STEPS]
SECTION: Creation of MAP2 surface
1. Add new surface.
Leave Creation Parameters as default
Selected segment only ROI.
Click next. Drag the yellow box to select the cell. Whole cell of interest.
Click Next. Select the channel (for your marker of interest). Smooth Surface detail. 0.0360. Machine learning segmentation.
Click Next. Draw thresholds; foreground and background w Shift-Click > 3 planes. Ensure the “pointer selection mode” is selected. 
Click Next. twice.
Threshold to have only one mask included.
Click Finish.
Go to Statistics, detailed tab, average values, save/export data.
        o  Save with suffix Cell #_MAP2
SECTION: Create LAMP1 Surface
2. Add new surface.
Add LAMP1 parameters. Ensure Select only a ROI is ticked. Ensure object-object stats is checked.
Click Next.
Drag yellow box to the ROI. Click Next.
Select green (LAMP1) channel. Smooth 0.0361. Machine learning selected. Click Next.
Add Machine Learning Thresholds. Foreground (green) and background (purple) w Shift-Click > 3 planes. Click Next.
Check enable. Seed Point Diameter 0.181.
Click Next. Adjust slide bar to highlight LAMP1 included regions. ClickNext.
Click Next. again and adjust.
Click Finish.
Remove LAMP1 regions by selecting the pencil (edit) and click the regions outside the cell and
‘delete’. Move through all the planes (z-axis).
Click Statistics, detailed tab; Average Values and export data.
        o  Save with suffix Cell #_LAMP1
SECTION: Create Cathepsin Spots
3. Create new ‘Spots’ for Cathepsin. Check the ‘different spot size (region growing). Click next.
Select ROI using the yellow box. Click Next.
Select the correct channel (red). Model XYZ diameter= 0.291, Estimated z diameter = 0.830. Check
background subtraction. Click Next..
Select threshold slider of Cat+ spots. For simplicity, select slice rendering for 3D spots. Click Next.
Check absolute intensity. Click Next.
Click Finish.
Remove Cathepsin dots outside the cell by selecting the pencil (edit) and click the regions outside the cell and ‘delete’. Move through all the planes (z-axis).
Click Statistics, detailed tab; Average Values and export data.
            o  Save with suffix Cell #_CatD
SECTION: Determining CatD-LAMP1 coalescence
4. Select filer. Add Shortest distance to LAMP1 surface #.
Slide scale bar to remove spots that aren’t touching.
Click duplicate selection.
Click Statistics, detailed tab; Average Values and export data.
            o  Save with suffix Cell #_CatD_co
TITLE: Genotyping mice from ear clips
AUTHORS: Elizabeth P.

[DESCRIPTION]
This is a protocol that describes the genotyping procedure from ear clip samples. This includes a general PCR protocol for primers with annealing temperatures in the range 55-70 degrees C. For other primers, the thermal cycling should be adjusted.

[STEPS]
SECTION: Tissue Collection
1. Scruffing mouse securely, snip a tissue sample approximately 4mm2 in size from the edge of its ear using a punch or scissors.
SECTION: Tissue Collection
2. Store and transport samples separately in clearly-labelled 1.5mL tubes.
SECTION: DNA Extraction
3. Add 200uL of 20mM NaOH to each tissue sample.
SECTION: DNA Extraction
4. Heat samples to 100 degrees C for 15 minutes, or until tissues are thoroughly dissolved.
SECTION: PCR amplification
7. Create master mix. Scale depending on how many reactions you are conducting.

SECTION: DNA Extraction
5. Add 50uL of 30% TRIS to each sample and centrifuge on high speed for 6 minutes.
SECTION: DNA Extraction
6. Withdraw 200uL of supernatant from each sample, store in 1mL tube. Use 1uL of this DNA solution for each reaction.
SECTION: PCR amplification
7.1. To create the mix, for each reaction add in order:
- 17 uL of autoclaved water
- 5 uL of GoTAQ buffer
- 0.5uL of dNTP
- 0.5uL of each primer
- 0.5uL of TAQ
SECTION: PCR amplification
8. Shake gently and add 24uL of this solution to DNA in a PCR plate.
SECTION: PCR amplification
9. Thermal cycle; the PCR protocol will vary according to primers, but cycling between 60 and 94 degrees Celsius has been effective for our lab.
SECTION: Gel electrophoresis
10. Prepare gel:
SECTION: Gel electrophoresis
10.1. - Heat 3g of agarose in 200mL TAE buffer solution.
- Once cooled to 55 degrees C, add 12uL SYBR Safe DNA Gel Stain
- Pour the gel onto the mold, add combs, and allow it to solidify for 15 minutes.
SECTION: Gel electrophoresis
11. Place solid gel into the electrophoresis chamber. Load PCR product from the plate into the wells, one per well, leaving a gap between each well. Add a ladder before the first and after the last well, or as desired.
SECTION: Gel electrophoresis
12. Switch on the electrical current (around 90 volts is recommended). Run the gel for 15-30 minutes depending on desired band size.
SECTION: Gel electrophoresis
13. Remove the gel and image. Using an Anxygen gel reader is recommended.