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

[DESCRIPTION]
<p>This is our ‘one-pot ligation’ protocol for Oxford Nanopore ligation libraries. It benefits from increased recovery of library and faster preparation time by using the Ultra II ligation module in conjunction with the Ultra II end repair/dA-tailing module therefore removing a clean-up step. It can be used with or without the optional FFPE DNA repair step which shoudl help to improve read-lengths in nicked DNA. The incubation times given here are sufficient to generate high quality libraries in under one hour. In the past we have cut these times in half without noticably impacting performance but we will leave this up to you.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Mitogenome Assembly with GetOrganelle
AUTHORS: Dakota Betz

[DESCRIPTION]
Basic instructions for assembling mitogenomes with the GetOrganelle Program

[STEPS]
SECTION: Installation
1. 
 

Before installation, activate your python environment (example code below for python3 environment):

 

SECTION: Installation
2. Install GetOrganelle with the following code:

 

You may need to install updates, be sure to say yes (type y and hit enter) when prompted to update any conda-related programs.

 

SECTION: Initialization
3. Next we need to download and initialize the database of your preferred organelle genome type.
Options include: embplant_pt, embplant_mt, embplant_nr, fungus_mt, fungus_nr, animal_mt, and/or other_pt

For animal mitochondrial genomes, use animal_mt:

 
If you would like to download and initialize multiple organelle genome types, you can list them after the --add  command above, separated by spaces.

 

SECTION: Initialization
4. If you have trouble with the above command (connection to GitHub fails) try a few times more, as the problem generally resolves itself. If you're still having trouble, you can download the repository  manually, as in Option 2 available here: https://github.com/Kinggerm/GetOrganelle/wiki/Initialization#default-directory
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient DNA extraction and purification of ancient bones.</p>

[BEFORE_START]
<p>Clean</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Agarose Gel Electrophoresis and Size-Selection of Library
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

[STEPS]
?. Prepare 3% low-melting temp agarose gel with 1:10,000 SybrSafe in 1× TBE.
?. [Prepare Samples and Run Gel]
Add  to each sample (18 μL of sample), mix.
[6× Orange G buffer]
?. [Prepare Samples and Run Gel]
Load on gel, leave one empty well between samples, 50 bp ladder on both sides of the gel.
?. [Prepare Samples and Run Gel]
Run ~95 V for  (longer gives better resolution but larger cut sizes).
?. [Gel-Extract Library from Gel]
Under blue light illumination, cut gel slice from 175–350 bp and place into 15 mL conical tube.
?. [Gel-Extract Library from Gel]
Use fresh razor blades for each sample and keep cross-contamination to minimum.
?. [Gel-Extract Library from Gel]
Cut and elute gel using Qiagen MinElute gel extraction kit:
?. [Gel-Extract Library from Gel]
Add 6× volumes of Buffer QG to melt gel (e.g., for 100 mg gel, add 600 μL QG).
?. [Gel-Extract Library from Gel]
Melt gel at  (do not heat) on benchtop (can shake to help melt, but do not vortex).
0 Room temperature
?. [Gel-Extract Library from Gel]
Add 1× volume of original gel of isopropanol and mix well (100 mg gel = 100 μL isopropanol).
?. [Gel-Extract Library from Gel]
Load on column (750 μL per spin, can do multiple spins, all spins max speed 1 min).
?. [Gel-Extract Library from Gel]
After all sample has been spun through, wash 1× with .
[Buffer QG]
?. [Gel-Extract Library from Gel]
Add 1× with , spin , pour out flow-through, spin again  max speed.
[Buffer PE]
?. [Gel-Extract Library from Gel]
Carefully move column to a new 1.5 mL tube, and air dry for .
?. [Gel-Extract Library from Gel]
Carefully add  directly to the center of the column, incubate for  at , spin at max speed.
[Buffer EB]
0 Room temperature
TITLE: 2-step PCR mixture and conditions (Barcoded-head primers for seqs pooling)
AUTHORS: Yin-Tse Huang, Tsu-Chun Hung

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
1. Wear glove, clean up the working bench w. 1% bleach
SECTION: For 1&#x27; PCR head-primers
2. Prepare 1' PCR master mixutre for head-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for head-primers for each reaction
 

 

SECTION: For 1&#x27; PCR head-primers
3. Mix the 1' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 1&#x27; PCR head-primers
4. Transfer  23.75 µL 1' PCR master mixutre in 8-strip PCR tubes.

SECTION: For 1&#x27; PCR head-primers
5. Add 1.25 µLDNA template in 8-strip PCR tubes, resulting in a 25 µL reaction mixture for 1' PCR.


 

SECTION: For 1&#x27; PCR head-primers
6. Mix the reaction mixture gently by tapping the tubes. Quick spin the tubes.
SECTION: For 1&#x27; PCR head-primers
7. Carry out PCR using the following condition:  

1' PCR condition for head-primers
 

SECTION: For 1&#x27; PCR head-primers
8. Carry out electrophoresis for inspection of DNA products

   

SECTION: For 1&#x27; PCR head-primers
9. Markdown wells and upload the pictures to the Lab Google drive

  

SECTION: For 2&#x27; PCR barcoded-head primers
10. Prepare 2' PCR master mixutre for barcoded-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for barcoded-primers for each reaction (NO PRIMERs at this point!!)
 

 

SECTION: For 2&#x27; PCR barcoded-head primers
11. Mix the 2' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 2&#x27; PCR barcoded-head primers
12. Transfer 13.05 µL of the 2' PCR master mixture to 8-strip PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
13. Add 1.2 µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
14. Add 0.75 µL of 1' PCR product as template, resulting in 15 µL reaction mixture for 2' PCR.

Negative control contains only 14.25 µL master mixture and premixed barcoded-head primers but not DNA template
SECTION: For 2&#x27; PCR barcoded-head primers
15. Mix gently by tapping the tubes. Quick spin the tubes.
SECTION: For 2&#x27; PCR barcoded-head primers
16. Carry out 2' PCR using the following condition:  

2' PCR condition for barcoded-head primers
 

SECTION: For 2&#x27; PCR barcoded-head primers
17. Carry out electrophoresis for inspection of DNA products

  

SECTION: For 2&#x27; PCR barcoded-head primers
18. Markdown wells and upload the pictures to the Lab Google drive
TITLE: Generation of Gba L444P mutant mouse
AUTHORS: Tae-Un Han, Edward Ginns

[DESCRIPTION]
This protocol was used to generate of mutant mouse carrying Gba L444P point mutation.
The protocol was also described in a previous literature (Mol Genet Metab. 2014 Feb;111(2):152-62. doi: 10.1016)

[STEPS]
1. The replacement targeting vector enabling positive/negative selection contained a neomycin resistance (neoR) cassette flanked by loxP sequences inserted into the intergenic regions between murine metaxin (mtx) and glucocerebrosidase (gba).
2. The L444P was introduced into a genomic clone of murine gba by PCR mutagenesis. For this, the normal murine Gba sequence in exon 9 was changed from TGACTTGGA to TGACCCGGA, resulting in the amino acid substitution of proline for leucine and introducing a Nci1 restriction site. Then, the change in sequence was confirmed by restriction digest and/or direct sequence analyses.
3. The final constructs contained a 4.0 kb 5′ gba homologous arm and a 1.4 kb 3′ mtx homologous arm.
4. A diphtheria toxin A (DTA) cassette was placed downstream as a negative selectable marker.
5. After linearization, the constructs were introduced into murine embryonic stem (ES) cells by electroporation, and the ES cells were subjected to drug selection in culture with G418 solution.
6. The correct gene targeting event in G418 resistant individual clones was identified by Southern blot and PCR analysis.
7. Cells from ES clones containing the correctly targeted mutation in one allele of the gba gene were injected into blastocysts from C57BL/6 mice and then transferred to foster mice.
8. Male offspring from these injections having more than 30% coat color chimerism were test-bred against C57BL/6 females, and progeny were screened by PCR and Southern analyses for transmission of the mutant gba allele.
9. Lines of mice containing the mutant gba allele were identified, and the DNA sequence confirmed by direct sequencing of PCR amplified DNA containing the mutation.
10. Mice heterozygous for the mutant gba gene were mated and homozygous mutant progeny were identified by Southern blot and PCR analysis.
11. In addition, heterozygous mice were mated to mice carrying a transgene for CRE DNA recombinase, resulting in the excision of the neoR marker, leaving only a 34 bp loxP sequence.
12. As expected, the targeted mutations were transmitted in a Mendelian fashion.
13. Mice homozygous for the gba mutations without the neoR cassette were used to expand the colonies.
TITLE: Electrophysiological studies
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo, Mikio Hayashi

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

[STEPS]
?. MIN6 cells were incubated in an extracellular bath solution containing 2 mM glucose for 30 min at 37 °C before patch-clamp experiments.
?. Membrane potential and whole-cell current were recorded using the EPC 800 patch-clamp amplifier (HEKA Elektronik Inc. Holliston, MA, USA). The amplifier was driven by Clampex 9 (Axon Instruments, Union City, CA, USA) to allow the delivery of a voltage-step protocol with concomitant digitization of the current. The capacitance transient current was compensated by the amplifier. Patch pipettes (G-1.5; Narishige, Tokyo, Japan) had a resistance of 5–7 MΩ when filled with the pipette solutions.The membrane potential was corrected for the liquid junction potential at the tip of the patch pipette in the KRBH buffer and for that at the tip of the indifferent reference electrode filled with KRBH buffer and placed in the bath.Whole-cell capacitance and series resistance (Rs) were 7.4 ± 1.5 pF and 13.3 ± 1.3 MΩ (n = 6), respectively.The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs. Patch pipettes (G-1.5; Narishige, Tokyo, Japan) had a resistance of 5–7 MΩ when filled with the pipette solutions.The membrane potential was corrected for the liquid junction potential at the tip of the patch pipette in the KRBH buffer and for that at the tip of the indifferent reference electrode filled with KRBH buffer and placed in the bath.Whole-cell capacitance and series resistance (Rs) were 7.4 ± 1.5 pF and 13.3 ± 1.3 MΩ (n = 6), respectively.The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs. The membrane potential was corrected for the liquid junction potential at the tip of the patch pipette in the KRBH buffer and for that at the tip of the indifferent reference electrode filled with KRBH buffer and placed in the bath.Whole-cell capacitance and series resistance (Rs) were 7.4 ± 1.5 pF and 13.3 ± 1.3 MΩ (n = 6), respectively.The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs. Whole-cell capacitance and series resistance (Rs) were 7.4 ± 1.5 pF and 13.3 ± 1.3 MΩ (n = 6), respectively.The Rs was not electronically compensated during the experiments, and the potentials reported here have not been corrected for the Rs.
?. The whole-cell current was filtered at 1 kHz with an internal four-pole Bessel filter, sampled at 2 kHz, and transferred to digital signals through Digidata 1322A (Axon Instruments).
?. A subsequent current analysis was performed using Clampfit 9 (Axon Instruments).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We present a lab-in-the-field experiment to demonstrate how individual behavior in the lab predicts their ability to identify phishing attempts. Using the business and finance staff members from a large public university in the U.S., we find that participants who are intolerant of risk, more curious, and less trusting commit significantly more errors when evaluating interfaces. We also replicate prior results on demographic correlates of phishing vunerability, including age, gender, and education level. Our results suggest that behavioral characteristics such as risk attitude intolerance to risk, curiosity, and trust can be used to predict individual ability to identify phishing interfaces.</p>

[STEPS]
TITLE: 1 Preparation of Mica Substrate
AUTHORS: Philip J. Haynes, Kavit H. S. Main, Alice Pyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 1 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]
?. [Preparation of Mica Substrate]
Cut the adhesive PTFE into circles of the same size as the steel sample discs (15 mm), using either a punch or scalpel (see Note 7).
?. [Preparation of Mica Substrate]
Peel off the backing of the PTFE cut-out and adhere to the steel disc.
?. [Preparation of Mica Substrate]
Mix the Araldite® 2-part epoxy resin 50:50 on a disposable surface e.g. weighing boat. Using a pipette tip transfer a small amount of the mixed epoxy to the centre of the teflon (use tip as a capillary to help bring up the glue).
?. [Preparation of Mica Substrate]
Cleave the 6 mm mica disc on one side with scotch tape. With the cleaved mica disc facing down, immediately place on top of the epoxy droplet and press flat.
?. [Preparation of Mica Substrate]
Leave the glue to dry and cure .
?. [Preparation of Mica Substrate]
Once cured, cleave the mica using Scotch tape to reveal an atomically flat clean substrate (see Note 8).
TITLE: Hematoxylin and Eosin (H&amp;E) Staining
AUTHORS: Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Hematoxylin and Eosin (H&E) Staining for histological sections

[STEPS]
SECTION: DEPARAFFINIZE
1. Xylene - 5 minutes

SECTION: HYDRATE
3. 100º alcohol - 6 quick immersions
SECTION: DEPARAFFINIZE
2. Xylene - 6 quick immersions
SECTION: HYDRATE
6. 70º alcohol - 6 quick immersions
SECTION: HYDRATE
5.  96º alcohol - 6 quick immersions
SECTION: HYDRATE
4. 100º alcohol - 6 quick immersions
SECTION: HYDRATE
7. 50º alcohol - 6 quick immersions
SECTION: HYDRATE
8. Distilled water (H₂O) - 2 minutes
SECTION: STAINING
9. Harris Hematoxylin - according to preparation
SECTION: STAINING
10. Running tap water (H₂O) - 5 minutes
SECTION: DIFFERENTIATE HEMATOXYLIN
11. Acid alcohol - 2 minutes. Acid alcohol is made by adding 0.3 ml of HCl to 250 ml of 70º alcohol.
SECTION: DIFFERENTIATE HEMATOXYLIN
12. Running tap water (H₂O) - 5 minutes
SECTION: BLUEING
13. NaHCO₃ - 1 minute. Add one tablespoon of NaHCO₃ to 250 ml of distilled water (H₂O).
SECTION: BLUEING
14. Running tap water (H₂O) - 5 minutes
SECTION: COUNTERSTAINING
15. 96º alcohol - 3 minutes. 
SECTION: COUNTERSTAINING
16. Eosin - according to preparation
SECTION: DEHYDRATE
17. 96º alcohol - 6 quick immersions
SECTION: DEHYDRATE
18. 96º alcohol - 6 quick immersions
SECTION: DEHYDRATE
19. 100º alcohol - 6 quick immersions
SECTION: DEHYDRATE
20. 100º alcohol - 6 quick immersions
SECTION: CLEAR
21. Xylene - until the preparations are clear. Leave in xylene until mounting.
TITLE: Intracellular and extracellular MHC-II immunostaining and microscopy on peritoneal macrophages
AUTHORS: Rebecca Wallings

[DESCRIPTION]
Staining of intracellular and extracellular MHCII in peritoneal macrophages in 96 well plate using EVOS fluorescent microscope and analysis on CellProfiler

[STEPS]
1. After harvesting and plating pMacs from mouse, plate in 96-well plate at 50,000 cells/well in 200uL as described in pMac harvesing protocol. Once cells have adhered, aspirate media and replace with fresh growth media containing vehicle or 100U of IFNy for 18
2. Retrieve cells from 37 degrees. Aspirate media and wash 3x DPBS+/+ 
3. Incubate cells with 25 ug/mL of APC-MHC-II (Biolegend, RRID:AB_313329) in DPBS+/+ containing 1:100 FcR blocking reagent, final volume 50uL, for 30 minutes at room temperature, protected from light
4. Wash cells 3 x DPBS+/+
5. fix cells by incubation in 4% PFA for 10 minutes at room temperature, final volume of PFA 50uL, and then washed 3x with DPBS+/+
6. Permeabilize cells with 100uL permeabilization buffer (eBiosciences, #88-8824-00) on ice for 15 minutes
7. Spike in 25ug/mL of PE-610-MHC-II (Biolegend, RRID:AB_2574618) into permeabilization buffer and incubate cells for 30 minutes at room temperature protected from light. 
8. Wash cells 3 x DPBS+/+
9. Incubate cells in 1 μg/ml DAPI (Invitrogen, RRID:AB_2629482) for 10 minutes at room temperature in DPBS+/+, final volume 50uL.  
10. Image cells on an EVOS‱ M7000 (Invitrogen) at 20 x magnification. 
11. Perform image analysis using Cellprofiler 4.2.5 (RRID:SCR_007358). Using the ‘IdentifyPrimaryObject’ module in Cellprofiler, identify icMHC and exMHC  in their respective channels and quantify MFI and calculate Ex:IcMHCII from these quantified MFI values.
TITLE: Do, Q. B. et al. (2023) Early deficits in an in vitro striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation
AUTHORS: Quyen Do, Humaira Noor, Ricardo Marquez Gomez, Kaitlyn M L Cramb, Bryan Ng, Ajantha Abbey, Naroa Ibarra-Aizpura, Maria-Claudia Caiazza, Parnaz Sharifi, Charmaine Lang, Dayne Beccano-Kelly, Jimena Baleriola, Nora Bengoa-Vergniory, Richard Wade-Martins

[DESCRIPTION]
This collection contains ten protocols detailing methods used in Do, Q. B. et al. (2023) Early deficits in an in vitro striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation. 

[STEPS]
TITLE: Making LB Agar Plate
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to make LB Agar Broth plates for bacterial quantification.</div><div class = "text-block"><span>*Protocol image used is from Wikimedia commons [</span><a href="https://commons.wikimedia.org/wiki/File:LB_agar_plate.jpg" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://commons.wikimedia.org/wiki/File:LB_agar_plate.jpg</span></a><span style = ":;">]</span></div></div>

[STEPS]
?. [Mix Broth]
Add  Miller's LB Agar Powder and  agarose to  Deionized Water.
2.5 g
1.5 g
100 ml
?. [Mix Broth]
Swirl powder in DIW until dissolved.
?. [Sterilize Broth]
Cover top of glassware containing broth solution with aluminum foil, and add a small piece of thermal indicator tape.
?. [Sterilize Broth]
Autoclave at  for  .
121 °C
Do not use the "Exhaust" mode on the autoclave as decreasing the pressure so suddenly will result in rapid boiling of solution. Allow the pressure to drop down on its own after the cycle is complete.
?. [Pour Plates]
Remove broth from autoclave while still hot.
?. [Pour Plates]
With one hand, move petri dish lid just enough to pour the broth.
?. [Pour Plates]
Pour broth into dish so that there is 5-7.5 mm of broth at the bottom.
?. [Store Plates]
Wrap edge of plate with parafilm to protect from contamination.
?. [Store Plates]
Once broth is firm enough, invert so condensation does not drip onto the medium.
?. [Store Plates]
Store plates in a plastic bag at  if they will not be used immediately.
4 °C
TITLE: H&E Staining for Pancreas Cryosections
AUTHORS: Diane Saunders, Angela Kruse, Jamie Allen, Carrie  Romer, Danielle Gutierrez, Alvin Powers, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.</div></div>

[STEPS]
?. Air dry sections for  .
?. Incubate in 95% alcohol for  , then wash, dipping until clear.
?. Incubate in hematoxylin for  , then wash until clear.
?. Dip 3-4x in bluing solution, then wash for
?. Dip 3-4x in 95% alcohol.
?. Dip 1-2x in eosin.
?. Dip 5-10x in 95% alcohol, then repeat using 2nd aliquot.
?. Dip 5-10x in 100% alcohol, then repeat using 2nd aliquot.
?. Dip 5-10x in xylene, then repeat using 2nd aliquot.
?. Apply coverslip and seal.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the suggested protocol for clean-up using AMPure XP beads. It is recommended as part of the <a href="https://www.protocols.io/view/jetseq-dna-library-preparation-kit-fwgbpbw" target="_blank">JetSeq<sup>TM</sup> DNA Library Preparation Kit protocol</a> for post ligation, post adaptor extenstion (PCR1), and post indexing clean-ups.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>MATERIALS:</strong><br /><br /> 1) NC64A <em>chlorella</em>, pelleted (fresh cells preferred)<br /> 2) 70% methanol<br /> 3) Centrifuge tubes, corex or polypropylene<br /> 4) 50 mM Tris-HCl, pH 7.8

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.