eamag/protocols-with-steps huggingface dataset:
TITLE: Homology Modelling of Pacer RH domain in SWISS-MODEL
AUTHORS: Dan Tudorica

[DESCRIPTION]
Using the Rubicon RH - Rab7 crystal structure as a template (6wcw)

[STEPS]
SECTION: Swiss-M
1. In Swiss Model,

> start new modelling project
> upload target sequence. Here, used the human Pacer (RUBCNL) sequence acquired from uniprot:

>sp|Q9H714|PACER_HUMAN Protein associated with UVRAG as autophagy enhancer OS=Homo sapiens OX=9606 GN=RUBCNL PE=1 SV=3
MVSQSTVRQDSPVEPWEGISDHSGIIDGSPRLLNTDHPPCQLDIRLMRHKAVWINPQDVQ
QQPQDLQSQVPAAGNSGTHFVTDAASPSGPSPSCLGDSLAETTLSEDTTDSVGSASPHGS
SEKSSSFSLSSTEVHMVRPGYSHRVSLPTSPGILATSPYPETDSAFFEPSHLTSAADEGA
VQVSRRTISSNSFSPEVFVLPVDVEKENAHFYVADMIISAMEKMKCNILSQQQTESWSKE
VSGLLGSDQPDSEMTFDTNIKQESGSSTSSYSGYEGCAVLQVSPVTETRTYHDVKEICKC
DVDEFVILELGDFNDITETCSCSCSSSKSVTYEPDFNSAELLAKELYRVFQKCWILSVVN
SQLAGSLSAAGSIVVNEECVRKDFESSMNVVQEIKFKSRIRGTEDWAPPRFQIIFNIHPP
LKRDLVVAAQNFFCAGCGTPVEPKFVKRLRYCEYLGKYFCDCCHSYAESCIPARILMMWD
FKKYYVSNFSKQLLDSIWHQPIFNLLSIGQSLYAKAKELDRVKEIQEQLFHIKKLLKTCR
FANSALKEFEQVPGHLTDELHLFSLEDLVRIKKGLLAPLLKDILKASLAHVAGCELCQGK
GFICEFCQNTTVIFPFQTATCRRCSACRACFHKQCFQSSECPRCARITARRKLLESVASA
AT
SECTION: Swiss-M
2. > Search for template
SECTION: Swiss-M
3. Search should yield a top hit corresponding to the Alphafold model of Pacer, and subsequent hits represent using the Rubicon RH domain as a template (6wcw).

Select the hit that aligns Pacer to the Rubicon structure (6wcw), and > build model. From here, the file can be downloaded as a PDB and used in downstream modelling steps.
SECTION: Build Rab7 complex model
4. >Open Pymol
>open downloaded Pacer homology model
>fetch rubicon-rab7 dimer crystal structure by typing "fetch 6wcw" in the command line
SECTION: Build Rab7 complex model
5. >Align Rubicon crystal structure and Pacer homology model using the "align" command.
SECTION: Build Rab7 complex model
6. >Delete rubicon monomer, and expansion binding site between the pacer homology model and rab7. Focus on S72 of Rab7
TITLE: HuBMAP: Paraffin Embedding Tissue Samples 
AUTHORS: Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the paraffin embedding of HuBMAP specimens.</div></div>

[STEPS]
?. [Post-Processing Tissue Acquisition]
Place processed cassettes in wax holding chamber of embedding center.Stock station with embedding utensils, gauze, and disposable embedding molds.
?. [Post-Processing Tissue Acquisition]
View the tissue sample and select an appropriately sized disposable embedding mold.
?. [Post-Processing Tissue Acquisition]
Using forceps, tap the dispenser plate on the embedding center's dispensing nozzle and partially fill a mold with  molten paraffin.
?. [Post-Processing Tissue Acquisition]
Using heated forceps, carefully transfer the tissue inside the cassette into the mold, placing the tissue cut side down (surface viewed when opening the cassette).
?. [Post-Processing Tissue Acquisition]
Open the cassette carefully maintaining tissue orientation. Dispose of cassette lid.
?. [Post-Processing Tissue Acquisition]
Preheat embedding utensils in the forceps warming chambers  located on the sides of the  paraffin dispensing nozzle.Turn on station cold plate module. .
?. [Post-Processing Tissue Acquisition]
Transfer the mold to the cold pad in front of the dispensing nozzle, and gently press tissue flat with forceps or a tamper.Allow paraffin to solidify in a thin layer, to hold the tissue in position.
?. [Post-Processing Tissue Acquisition]
Once oriented with the tissue specimen in the center of the mold, place the lower basket of the emptied cassette on top of the mold.
?. [Post-Processing Tissue Acquisition]
Add additional molten paraffin to 2/3 fill the cassette basket.Remove any bubbles that may form between the mold and the cassette by slowly lifting and repositioning the cassette.
?. [Post-Processing Tissue Acquisition]
Carefully transfer paraffin filled mold to freezing/cold plate module of embedding station. Allow paraffin to completely solidify.
?. [Post-Processing Tissue Acquisition]
If you have additional tissue samples to embed, return to step 4 and repeat through step 11.
[if you have additional samples.]
TITLE: Ketosium ACV Gummies Reviews &amp; Shark Tank, Where To Buy
AUTHORS: Ketosium  Reviews

[DESCRIPTION]
Ketosium ACV Gummies Reviews & Shark Tank, Where To Buy

[STEPS]
SECTION: Ketosium ACV Gummies Reviews &amp; Shark Tank, Where To Buy Keeping one’s body in tip-top shape can be grueling, and numerous people can not do so. It&#x27;s getting decreasingly delicate to maintain a healthy life in the current world. There are numerous reasons why someone might not be in peak physical condition. There&#x27;s a result that could break this problem without farther oil.   Ketosium ACV Gummies is your stylish chance right now if you ’re looking to slim down and lose weight naturally. It’s a natural supplement that uses the ketosis process to burn fat. This Keto diet plan is a important or popular way to lose weight these days.   What&#x27;s Ketosium ACV Gummies   To increase the body’s natural product of beta-hydroxybutyrate, the manufacturer created Ketosium ACV Gummies, a keto supplement. For those who need a little fresh help burning fat, this supplement comes in the shape of capsules. By boosting metabolism and burning fat to energy in your system, this supplement reviews claim that it aids in weight loss. BHB ketones, added to this supplement blend, increase energy and fat burning while dwindling fat storehouse.   Advanced Appetite Enhancing Components   Cutting- edge factors and a potent weight loss drug can help people reach their ideal weight in a short quantum of time. The results of the herbal remedies will be known during this period.   Apple Cider Ginger   The ginger’s sour, smelling flavor is deduced from the apple juice employed in its product. To give color- treated hair some redundant radiance, apply it to the ends of your hair as an antibacterial treatment. With the right combination of chemicals, this material can be converted into colorful useful products (and perhaps indeed some taste tests).   Official Website:- https://ipsnews.net/business/2022/05/01/ketosium-acv-gummies-reviews-does-it-have-side-effects/
1.
TITLE: Fontana-Masson staining
AUTHORS: Cristian González-Cabrera, Csilla Novák, Andrés Mauricio Jaramillo Flautero, Celine Winter, Matthias Prigge

[DESCRIPTION]
The Fontana-Masson Staining Protocol is a detailed method for staining free-floating sections (20-50 microns) to detect melanin and other pigments in tissue samples. The procedure involves preparing a silver nitrate working solution, treating sections with triton, and sequential incubation in pre-heated silver nitrate solution, gold chloride, and thiosulfate solutions, with thorough washing steps between each incubation. The process concludes with sections being placed in PBS for mounting and counterstaining. This protocol is critical for histological studies in biology, particularly for pigment analysis in tissues.

[STEPS]
SECTION: Fontana-Masson staining
1. Use free floating sections between 20-50 microns.
SECTION: Fontana-Masson staining
2. Silver Nitrate working solution:
- Prepare a 1:3 solution of silver nitrate solution in distilled water. Prepare enough solution for the amount of sections you want to stain.
- Add drop by drop concentrated ammonium hydroxide, while shaking, until the solution gradually turns transparent. Be careful and stop the addition of ammoniacal solution as soon as you get transparency.


 

 

SECTION: Fontana-Masson staining
3. Place the sections to stain (up to 7-8 sections) in a 2 mL Eppendorf tube. Incubate the sections in 0.3% triton for two hours at room temperature while shaking

SECTION: Fontana-Masson staining
4. Wash 3 times with distilled water for 5 min each.
SECTION: Fontana-Masson staining
5. Pre-heat the silver nitrate working solution for 5 minutes at 60 °C.

SECTION: Fontana-Masson staining
6. Incubate the sections in the preheated solution for 20 minutes at 60 °C.

SECTION: Fontana-Masson staining
7. Wash the sections 3 times with abundant distilled water.
SECTION: Fontana-Masson staining
8. From now on, perform the following steps in individual sections:
SECTION: Fontana-Masson staining
8.1. Incubate the section with gold chloride solution. Apply enough solution to cover the section (approximately 100-150 µL). Manually shake the tube for 30 seconds.

SECTION: Fontana-Masson staining
8.2. Wash the section 3 times with abundant distilled water.
SECTION: Fontana-Masson staining
8.3. Incubate the section with thiosulfate solution. Apply enough solution to cover the section (approximately 100-150 µL). Manually shake the tube for 2 minutes.

SECTION: Fontana-Masson staining
8.4. Wash with tap water for 2 minutes.
SECTION: Fontana-Masson staining
8.5. Wash 3 times with distilled water for 5 min each.
SECTION: Fontana-Masson staining
9. Place the sections in PBS for mounting, counterstaining, etc.


SECTION: Results
10. Slices should now show an amplified pigmentation as show in figures
TITLE: DNA extraction and Nested PCR
AUTHORS: Baiyan Gong1 ¶, Yaming Yang2 ¶, Xiaohua Liu, Jianping Cao, Meng Xu, Ning Xu, Fengkun Yang, Fangwei Wu, Benfu Li, Aiqin Liu *, Yujuan Shen *

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol for the Nested PCR assay</div></div>

[STEPS]
?. DNA extraction
?. PCR amplificationAmplification was accomplished in final volume of 25 μL, containing 17 μL of DNase/RNase-Free Deionized Water, 0.5 μL of each primer, 0.5 μL of TaKaRa Taq DNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan), 2.5 μL of 10 × PCR Buffer, 2 μL of 10 mmol dNTP and 2 μL of DNA .
?. The target DNA undergoes the first run of PCR with the first set of primers.Initial denaturation
?. Denaturation
?. Annealing
?. Extension/elongation
?. DNA extractionGenomic DNA of E. bieneusi was  extracted directly from 180 to 200 mg of each fecal specimen using the recommended procedures and the provided reagents by the manufacture of QIAamp DNA Stool Mini Kit (QIAGEN,  Hilden, Germany). To obtain high yield of DNA, the lysis temperature was increased to 95 ° C according to the manufacturer’s suggestion. DNA was eluted in 200 μL of AE and stored at −20°C before it was used for PCR analysis.
?. [35 cycles]
?. Final extension
?. The product from the first reaction undergoes a second run of PCR with the second set of primers.Initial denaturation
?. Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR products.
?. All secondary PCR  products were subjected to electrophoresis in a 1.5 % agarose gel and visualized by staining the gel  with GelStrain (TransGen Biotech., Beijing, China).
?. Final extension
?. Extension/elongation
?. Denaturation
?. 
?. Annealing
?. Extension/elongation
?. [30 cycles]
?. Final extension
?. Final holdThe final step cools the reaction chamber to 4°C for an indefinite time, and may be employed for short-term storage of the PCR products.
?. All secondary PCR  products were subjected to electrophoresis in a 1.5 % agarose gel and visualized by staining the gel  with GelStrain (TransGen Biotech., Beijing, China).
TITLE: Tissue Preparation for CLARITY
AUTHORS: Seth Currlin, Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tissue preparation for CLARITY includes fixation in 4% PFA, infusion with monomers in Hydrogel Solution, and clearing of lipids in Electrophoretic Tissue Clearing Solution. </div></div>

[STEPS]
?. 1.	PFA Incubation: Trimmed tissues are placed fixed in 4% PFA for 20-24 hours at room temperature on a slow platform rocker.
?. 2.	Hydrogel Incubation: Tissues are transferred to Hydrogel Solution in for approximately 5 days at 4˚C. Tubes are kept on a 2-axis rocker for the duration.
?. 3.	Hydrogel Polymerization: On day 5 the tissue is placed in pre-warmed 37˚C PBS and a layer of mineral oil to deter oxygen exchange from air within tube. The hydrogel within the tissue is polymerized at 37°C for 4 hours with gentle rocking.
?. 4.	Passive Clearing: Following polymerization the tissue is placed in Clearing Solution and kept at 45˚C with gentle rocking until optically clear.
TITLE: U Mass - Creatine Kinase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Creatine Kinase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Collection of Mammalian Embryonic Palate Tissue
AUTHORS: Matthew Fox

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will guide you through the process of extracting embryos from adult mice and the manipulation of embryos in order to obtain palatal tissue.</div></div>

[STEPS]
?. [Preparation]
Euthanize pregnant mouse via primary and secondary modes of IACUC approved euthanasiaCO2 gas at 2L/min until 1 minute after last gasp.Cervical separation
?. [Embryo Collection]
Using scissors, create an abdominal incision from the genitalia to the rib cage.
?. [Embryo Collection]
Locate and lift both strands of embryos so they are clear of all organs.
?. [Embryo Collection]
Free embryos by cutting the tissues connecting anteriorly at the kidney and posterior at the base of the uterine horn.
?. [Embryo Collection]
Transfer the embryos to the 50ml tube containing PBS on ice.Embryos should remain on ice until all sample collection is complete.
?. [Embryo Collection]
Place adult mouse in ziplock bag and place in Animal Room freezer
?. [Processing]
In a dish containing PBS, remove each embryo from the uterine strand using micro scissors.
?. [Processing]
Cut open the amniotic sacs and cut the umbilical cords to free the embryos from the placenta.
?. [Processing]
Remove the heads of the embryos and transfer to a dish of clean PBS.
?. [Palate Collection]
Transfer an embryo head to a clean sorting dish and place under stereoscope at 1.75X zoom.
?. [Palate Collection]
Remove tongue and mandibular tissues.
?. [Palate Collection]
Excise palatal shelves via cutting the tissues connecting the palatal shelves to the nasal palatal and maxillary processes.
?. [Palate Collection]
Transfer palate tissue to 1.5ml centrifuge tube on ice.
?. [Preservation]
Preserve palate tissue by freezing at   in PBS solution.
-80 °C
TITLE: Protein extraction and Western blotting
AUTHORS: Shiyi Wang

[DESCRIPTION]
Protein extraction and Western blotting

[STEPS]
1. **Prepare Membrane Solubilization Buffer** - 25 mM Tris (pH 7.4) - 150 mM NaCl - 1 mM CaCl2 - 1 mM MgCl2 - 0.5% NP-40 - Protease inhibitors
2. **Wash Cultured Astrocytes** - Wash cultured rat astrocytes twice with ice-cold TBS containing 1 mM CaCl2 and 1 mM MgCl2.
3. **Extract Protein** - Incubate astrocytes on ice in membrane solubilization buffer for 20 minutes with occasional agitation.
4. **Collect Cell Lysates** - Vortex briefly. - Centrifuge at 4°C at high speed for 10 minutes to pellet non-solubilized material.
5. **Store Supernatant** - Collect the supernatant and store at −80°C.
6. **Determine Protein Concentration** - Use the Pierce BSA Protein Assay Kit (Thermo Fisher).
7. **Prepare Protein Samples** - Mix lysates with 4x Pierce™ Reducing Sample Buffer (Thermo Scientific). - Incubate at 45°C for 45 minutes to denature proteins.
8. **Load Protein into Gels** - Load 7-10 μg of protein (cultured astrocyte lysates) into Bolt™ 4–12% Bis-Tris Plus gels (Thermo Scientific). - Run at 150 V for 1 hour.
9. **Transfer Proteins to Membrane** - Transfer proteins at 100 V to PVDF membrane (Millipore) for 1.5 hours.
10. **Block Membrane** - Block in 5% BSA in TBST (137 mM NaCl, 2.68 mM KCl, 24.7 mM Tris-Base, 0.1% (w/v) Tween 20) for 1 hour.
11. **Incubate with Primary Antibodies** - Incubate overnight at 4°C with the following primary antibodies: - Anti-LRRK2 (Rabbit, 1:500; ab133474, Abcam) - GAPDH (Mouse, 1:5000; ab8245, Abcam) - β-actin (Mouse, 1:5000; A5441, Millipore Sigma)
12. **Wash Membranes** - Wash membranes with TBST.
13. **Incubate with Secondary Antibodies** - Incubate in HRP secondary antibodies (Thermo Fisher Scientific) for 2 hours.
14. **Wash and Image Membranes** - Wash membranes in TBST. - Image on a Biorad Gel Doc imaging system.
15. **Quantify Protein Levels** - Quantify protein levels using FIJI software.
TITLE: Uploading Data Files to Terra 
AUTHORS: Francis J Ambrosio

[DESCRIPTION]
Uploading data to Terra.bio is an essential step in the protocol for analyzing locally stored genomic sequencing data. The Terra.bio uploads page allows users to easily organize their data files using an associated metadata file via a browser-based graphical user interface. This protocol explains the process to prepare the data files and the associated metadata file for upload, and provides the link to the Terra.bio uploads page.

[STEPS]
SECTION: Upload Data Files
1. Navigate to the Terra.bio uploads page.
  

SECTION: Upload Data Files
2. Select an existing workspace or create the workspace where the data will be uploaded. If there are no workspaces created under the billing project associated with this account please reach out to support@theiagen.com and we will facilitate the creation of a workspace pre-loaded with workflows to perform the analyses needed to meet any variety of research objectives.
   

SECTION: Upload Data Files
3. Select an existing collection or create the collection where the data will be uploaded.
  

SECTION: Upload Data Files
4. Drag and drop files into the indicated region of the page (or use the file browser by selecting the plus button in the bottom right hand of the window).
   

SECTION: Upload Metadata File
5. Using the sample names and file names of the the files that have been uploaded to Terra, create a spreadsheet of this "metadata" to upload to Terra as well.

Cell A1 defines the root entity. This will be the name of the table where the data is stored in Terra. The root entity is defined using the "entity:" notation and must end with "_id".
  
 *NOTE: It is best practices to include the run_id of each sample in an addition column:
  

SECTION: Upload Metadata File
6. Save the file as a tab delimited text file:
  

SECTION: Upload Metadata File
7. Drag and drop the metadata file into the indicated region of the page (or use the file browser by selecting the plus button in the bottom right hand of the window).
  

SECTION: Upload Metadata File
8. Review the entries in the datatable and click 'CREATE TABLE' to complete the upload process
  
  
If you experience any issues during this process please reach out to our support email:
support@terrapublichealth.zendesk.com

If video is your preferred medium of support check out our youtube video on uploading paired end sequencing data to Terra.bio:
https://youtu.be/DTU4rON0ycI
TITLE: DAB-quant
AUTHORS: Sneh Patel, Sara Fridovich-Keil, Shauna Rasmussen, and Judith L Fridovich-Keil

[DESCRIPTION]
Here we describe DAB-quant, a new system that facilitates quantitation of large numbers of scanned tissue slides stained via immunohistochemistry with 3,3′-Diaminobenzidine (DAB). The python code, instructions, license, and a link to example scans for analysis are all available at: 
https://github.com/sarafridov/DAB-quant

(The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)

[STEPS]
SECTION: Protocol
1. Here we provide DAB-quant, a new system that facilitates quantitation of large numbers of scanned tissue slides stained via immunohistochemistry with 3,3′-Diaminobenzidine (DAB). The python code, instructions, license, and a link to example scans for analysis are all available at:
 https://github.com/sarafridov/DAB-quant
SECTION: Spotlight video
2.
TITLE: Preparing samples for NGS
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes a standard procedure used to prepare PCR samples for Next Generation Sequencing (NGS)



[STEPS]
2. Purify the rest of the PCR product using column based or SPRI-beads based method, like Wizard SV Gel and PCR clean-up system or AMPure XP beads 
3. Following the instruction of the NGS facility, dilute the purified amplicon to a proper concentration, usually 10 ng/µl 
4. Send the purified samples to a NGS facility for multiplexing, library prep and NGS.
1. Run 5 µl PCR product in a 1% agarose gel to confirm successful amplification
TITLE: TX-100 FRACTIONATION PROTOCOL
AUTHORS: Svermily  

[DESCRIPTION]
To isolate insoluble and soluble proteins from dissected brain regions frozen for biochemical analysis.

[STEPS]
SECTION: Thaw Tissue
1. If tissue stored at -80 °C, place in -20 °C for at least 240 min or 240 min to thaw prior to homogenization.

SECTION: Prepare 1X TNE
2. Prepare 1X TNE with phosphatase and protease inhibitors.
SECTION: Homogenize Tissue
3. Weigh tissue out in mg.
SECTION: Homogenize Tissue
4. Add in 10 volumes of 1X TNE.
10 µL of TNE per 1 mg of tissue. 
Ex: 50 mg tissue = 500 µL of 1X TNE.
SECTION: Homogenize Tissue
5. Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and on ice.
SECTION: Homogenize Tissue
6. This is TNE crude lysate (no detergents).
SECTION: Prepare for Soluble v Insoluble
7. Take specific volume of tissue in TNE and add in equal volume of 1X TNE w/ 2% Triton X-100 (Tx100).

Ex. 150 µL of TNE tissue + 150 µL 1X TNE+2% Tx100.

SECTION: Prepare for Soluble v Insoluble
8. Sonicate @ 4 °C. 
SECTION: Prepare for Soluble v Insoluble
9. Spin down. 
SECTION: Prepare for Soluble v Insoluble
10. After spin: 
SECTION: Prepare for Soluble v Insoluble
11. Wash pellet in 150 µL of 1X TNE+1% Tx100.
 


SECTION: Prepare for Soluble v Insoluble
12. Resuspend pellet in 75 µL to 100 µL of cTNE.

SECTION: Prepare for Soluble v Insoluble
13. Sonicate @ 4 °C: 3 pluses: 10 s ON / 2 s OFF.

SECTION: Prepare for Soluble v Insoluble
14. Boil: 10 min at 95 °C.

SECTION: Prepare for Soluble v Insoluble
15. Spin down: 16000 x g, 15 min, 4 °C.

SECTION: Prepare for Soluble v Insoluble
16. Supernatant from this is the insoluble fraction.
SECTION: Prepare for Soluble v Insoluble
8.1. 3 pulses:10 s ON / 2 s OFF.

SECTION: Prepare for Soluble v Insoluble
9.1. Option 1: 16000 x g, 15 min, 4 °C.

SECTION: Prepare for Soluble v Insoluble
9.2. Option 2: 20000 x g, 60 min, 4 °C.

SECTION: Prepare for Soluble v Insoluble
10.1. Supernatant = soluble. Save supernatant and add equal volume of complete TNE (cTNE). 

Sonicate @ 4 °C: 3 pluses: 10 s ON / 2 s OFF.
Boil: 10 min at 95 °C. 
Spin down: 16000 x g, 15 min, 4 °C. 
Supernatant from this is the soluble fraction.
SECTION: Prepare for Soluble v Insoluble
10.2. Pellet = insoluble.
SECTION: Prepare for Soluble v Insoluble
11.1. Resuspend pellet via pipette. 
SECTION: Prepare for Soluble v Insoluble
11.2. Spin down. 

Option 1: 16000 x g, 15 min, 4 °C. 
Option 2: 20000 x g, 60 min, 4 °C (25000 x g) .
TITLE: SMARTerV4 (0.5x) Amplification for single-cell or single-nuclei RNASeq Protocol
AUTHORS: Allen Institute

[DESCRIPTION]
Protocol to generate full-length cDNA from single cells, or nuclei, using Takara SMARTer V4.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was used to measure sealer penetration into dentinal tubules of the root canal and sealer penetration into the perimeter of the root canal walls, by mean of ImageJ program (version 1.51j8) in image samples obtained by means of confocal microscopy.</p>

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