eamag/protocols-with-steps huggingface dataset:
TITLE: HTTM : gDNA extraction
AUTHORS: Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue

[DESCRIPTION]
Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets. 

[STEPS]
SECTION: DNA extraction
2. Add 600 µLof lysis solution to each well of the deep-well plate and resuspend the pellet. 

SECTION: DNA extraction
1. Prepare the lysis solution by adding 165 µL of proteinase K to 66 mL of homemade lysis buffer and mix well. 

SECTION: DNA extraction
3. Cover with an adhesive aluminum foil and incubate at 55 °C for 60 min. 

SECTION: DNA extraction
4. While still warm, add 260 µLof ethanol 100%, without overmixing.

 

SECTION: DNA extraction
5. Transfer immediately to a deep-well plate fitted with an array of silica columns. 
SECTION: DNA extraction
6. Centrifuge twice at 3270 x g, 10 min . 


SECTION: DNA extraction
7. Discard flowthrough and add 500 µL of wash solution.

SECTION: DNA extraction
8. Centrifuge at 3000 x g, 10 min 


SECTION: DNA extraction
9. Discard flowthrough.

SECTION: DNA extraction
10. Centrifuge at 3000 x g, 5 min  to eliminate traces of wash solution. 
SECTION: DNA extraction
11. Discard flowthrough.

SECTION: DNA extraction
12. Add a collector plate between the silica column array and the deep-well plate. 
SECTION: DNA extraction
13. Add 50 µLof low TE to the silica matrix in each well. 

SECTION: DNA extraction
14. Cover with an adhesive aluminum foil and incubate at55 °C for 15 min.

SECTION: DNA extraction
15. Centrifuge at 3270 x g, 5 min .

SECTION: Silica array regeneration (Optional)
16. Put the contaminated silica array on an empty deep-well plate.
Add 150 µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well. 

SECTION: Silica array regeneration (Optional)
17. Incubate at Room temperature for 5 min

SECTION: Silica array regeneration (Optional)
19. Add 200 µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
18. Centrifuge 3000 x g, 2 min 

SECTION: Silica array regeneration (Optional)
20. Incubate at Room temperature for 30 min 

SECTION: Silica array regeneration (Optional)
21. Centrifuge 3000 x g, 2 min 

SECTION: Silica array regeneration (Optional)
22. Add 150 µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
23. Incubate at Room temperature for 5 min

SECTION: Silica array regeneration (Optional)
24. Centrifuge 3000 x g, 2 min 
SECTION: Silica array regeneration (Optional)
24.1. Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through. 
SECTION: Silica array regeneration (Optional)
25. Add 600 µLof ddH2O to each well. 

SECTION: Silica array regeneration (Optional)
26. Centrifuge 3000 x g, 5 min 
SECTION: Silica array regeneration (Optional)
27. Silica columns array are ready to be reused. 
SECTION: DNA extraction
8.1. Repeat steps 7 and 8.
TITLE: Soil pH 1:1 Soil Water Ratio 
AUTHORS: maggie.bowman

[DESCRIPTION]
Soil pH can be measured at either a 1:1 or 1:2 soil to water ratio. Because of the use of the log scale, the two ratios typically provide similar results. A soil with high clay or organic matter may require a 1:2 or even 1:5 ratio to allow for enough fluid to make a measurement. A sample that will also have the lime requirement (i.e. Sikora) measured will be measured with a 1:1 ratio when possible. With either soil to water ratio, the soil and water are placed in a tube and shaken for 15 minutes on a reciprocating shaker to achieve soil solution equilibrium. A calibrated probe accounting for temperature is used for the measurement. One CAL standard sample is included in each batch. 

[STEPS]
1. Measure 20 g dry weight soil into the Falcon tube (this should be done in the core processing step)

2. Add 20 mL 

3. Place on reciprocating shaker for 15 min at 1000 RPM

4. Rinse the pH probe well with DI water and gently pat dry with a kimwipe
5. Check the calibration of the pH meter using the ph 4.01 and 7 standard. If either of the pH values differ from the known calibrate the pH probe.
6. Insert rinsed pH/temperature probe combo into the sample.
7. Between each measurement, rinse the probe well with DI water over the beaker and gently pat dry the probe with a kimwipe.
8. When not measuring, make sure that the pH probe is rinsed and fully submerged in 3 Molarity (M).

9. After measuring the pH, discard the soil water slurry into the autoclave waste (smaller containers on the counter or large container under bench near the hood).
10. Print the required labels needed for Bray/Olsen extraction for each site, depth, and replicate. 
5.1. Calibrate the pH meter with a minimum of two standards that bracket the potential sample results according to the instructions on the screen.
5.2. The probe should be calibrated using standards in the same type of vessel as the samples will be measured in (centrifuge tubes).
6.1. Be careful to keep the probes still and not against the wall or bottom of the vessels 
6.2. Make sure the diaphragm is submerged in suspension. 
6.3. Record results when the numbers stabilize onto the pH log sheet.
Soil_pH_log.xlsx
10.1. 1000S_SITE_P_BRAY_DEPTH_REPLICATE or 1000S_SITE_P_OLSEN_DEPTH_REPLICATE
10.2. 1000S_SITE_Pext_BRAY_DEPTH_REPLICATE
TITLE: Purification of avian egg yolk immunoglobulins using the water dilution method, precipitation with isopropanol and using HiTrap™ Columns.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. Separate egg yolk from the egg white.
?. Add nine parts of distilled water to one part egg yolk.
?. Mix and stir slowly for 6.30 h at 4°C.
?. Centrifuge at 10 000 × g, at 4°C for 25 min to precipitate the lipids.
?. Collect the supernatant containing the IgY (water soluble fraction).
?. Slowly add 5 ml of cold isopropanol to the preparation, stirring constantly, and centrifuge at low spead.
?. Dilute the precipitate in PBS and adjust pH to 7.6. Calculate the protein concentration by ELISA. spectrophotometry or Bradford method.
?. Determine the purity of the preparation by SDS-PAGE.
?. Fill the syringe or pump tubing with de-ionized water. Remove the stopper and connect HiTrap™ column to syringe (use the connector supplied).
?. Snap off tab on the column outlet.
?. Wash out the ethanol with 26 ml of de-ionized water.
?. Equilibrate column with 26 ml of binding buffer. The recommended ﬂow rate is  5ml/min.
?. Apply the IgY sample using a syringe ﬁtted to Luer connector or by pumping it onto the column.
?. For better results, use a ﬂow rate of 0.5 to 5.1 ml/min during sample application.
?. Wash with at least 51 ml of binding buffer or no material remains in the efﬂuent.
?. Maintain a ﬂow rate of 5 to 11 ml/min for washing.
?. Elute with 51 ml of elution buffer using a one-step or using a linear gradient though larger volumes are often required to break the interaction.
?. After elution, regenerate the column by washing with 36 ml of wash buffer.
?. Re-equilibrate the column with 26 ml of binding buffer.
?. The column is now prepared for a new puriﬁcation.
TITLE: Pichia pastoris transformation through electroporation
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Prior]
Prior to transformation plasmid DNA containing the gene(s) of interest was linearized in restriction digests using an enzyme that only cuts once following the manufacturers manual !
?. [Transformation]
Mix approx. 150 ng of linearized vector DNA with electrocompetent Pichia cells
?. [Transformation]
Tranfer mixture to ice-cold 0.2 cm electroporation cuvette
?. [Transformation]
Incubate cuvette  for 2 min
?. [Transformation]
Electroporate using a BioRad MicroPulser with a charging voltage of 1.5 kV
?. [Transformation]
Immediately  add 1 mL of 1:1 1 M sorbitol and YPD (v/v)  to recover the cells
?. [Transformation]
Transfer mixture to a plastic culture tube and incubated at 30°C for 1 hour at 225 rpm.
?. [Transformation]
Centrifuge culture tube at 3.000 x g for 5 min to pellet the cells
?. [Transformation]
Resuspend pellet in 200 μL YPD media
?. [Transformation]
Plate out cells on YPDS plates containing desired antibiotic and incubated at 30°C for 3 days
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol, which was developed in 2006 by Santiago Perez while in the Weis lab, provides a way to temporarily immobilize small Aiptasia for live imaging on a confocal microscope. As written, the protocol is used for inverted confocal microscopes, but the glass-bottom dishes can be replaced with a normal glass slide and coverslip for an upright microscope.</p>

[GUIDELINES]
<p>NOTE: If you are going to take images that don’t require long scanning times (which would thus require well-immobilized anemones) skip embedding in agar and simply use the MgCl<sub>2</sub>/seawater medium.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Cas9 Nuclease, S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).

[BEFORE_START]
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found here.
Reactions are typically 30 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
It is essential to keep the molar ratio of Cas9 and sgRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here.
If planning to use higher concentration Cas9 Nuclease, S. pyogenes (NEB #M0386T and NEB #M0386M) for in vitro digestion of DNA, the enzyme can be diluted to 1 Micromolar (µM) in 1 X and used immediately.  If the 1 µM dilution will be stored at -20 °C, it should be diluted using Diluent B (NEB #B8002S): 300 Milimolar (mM), 10 Milimolar (mM), 0.1 Milimolar (mM), 1 Milimolar (mM), 500 μg/ml  and 50 % (pH 7.4 @ 25 °C) prior to the reaction assembly.

[GUIDELINES]
REFERENCES:
Jinek et al. (2012) Science 337 (6096) 816-821.
Larson et al. (2013) Nature Protocol 8 (2180-2196).
Mali et al. (2013) Science 339 (6121): 823-826.

[STEPS]
3. Assemble the reaction at 37 Room temperature in the following order:
 

4. Pre-incubate for 10 min at 25 °C.
5. Add 3 µL (3 nM final).
6. Mix thoroughly and pulse-spin in a microfuge.
7. Incubate at 37 °C for 15 min.
8. Add 1 µL to each sample. Mix thoroughly and pulse-spin in a microfuge.
9. Incubate at 37 Room temperature for 10 min.
10. Proceed with fragment analysis.
1. Prepare 300 Nanomolar (nM) by diluting the stock with nuclease-free water on ice.
2. Prepare 30 Nanomolar (nM) with a single target sequence by diluting the stock with nuclease-free water on ice.
TITLE: JAX - EZ Lysis Nuclei Isolation for 10x Genomics Assays
AUTHORS: William F F F. Flynn, Elise  Courtois, Greg  Greg Perry, sandy.daigle  , Paul.gabriel  , Diane  Luo, Jessica  Grassmann

[DESCRIPTION]
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the  10x Genomics Multiome assay.  

This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.

This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).

[BEFORE_START]
Familiarize yourself with 10x Genomics protocol CG000365.

[GUIDELINES]
Assess nuclear membrane integrity (refer to 10x Genomics CG000365), using Brightfield or Trypan blue
Assess for debris, if large amount of debris present, filter with smaller pore size to decrease debris. 
Use either Trypan blue and Countess (for clean average size nuclei preps) or AO/PI and Nexcellom K2 counter (if nuclei prep has debris)
If RNA content is of concern Imaging nuclei can be done with fluorescence, and Syto RNA stain


[STEPS]
SECTION: Sample Prep
1. Slightly thaw sample out on ice and place onto pre chilled Petri dish on ice.
SECTION: Sample Prep
2. Cut tissue into small pieces and add to a microcentrifuge tube.
SECTION: Cell lysis
3. In micro centrifuge tube containing the tissue add 100 µL of lysis buffer.

SECTION: Cell lysis
4. Using a plastic pestle to grind tissue by pushing pestle down and twisting to break up the tissue. 
Start timer once lysis buffer is added.
SECTION: Cell lysis
5. Add 25 µL to wash off pestle and place tube on ice for a total of 5 min.

SECTION: Cell lysis
6. After 5 minutes of lysis on ice, 500 x g, 5 min, 4 °C.

SECTION: Cell lysis
7. Remove supernatant and resuspend pellet using wide bore tips in 100 µL.

SECTION: Cell lysis
8. Incubate on ice for a total of 5 min.

SECTION: Washing
9. Add 500 µL and 500 x g, 5 min, 4 °C.

SECTION: Washing
10.   
SECTION: Washing
11. After last wash, filter suspension through 40um filter.
SECTION: Washing
12. Resuspend sample in 1x Nuclei buffer (10x provided) containing RNase inhibitor, and DTT.
SECTION: Load
13. Load for transposition and continue with the 10x multiome protocol.
TITLE: Corchea: paper-based microfluidic device
AUTHORS: Gonzalo Vidal, Tim Rudge

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Corchea is a paper-based microfluidic system for culture and comunicate bacteria on flow. Its has a hardware that you can make with  a 3D printer and laser cut. The system allows the directed comunications between biofilms and we aim to array biological logic gates on it to make complex information processing.  </div></div>

[STEPS]
?. [Paper design]
Draw the borders of your device and the future channels in your paper (#1 Whatman)
?. [Paper design]
Cut the borders
?. [Paper design]
Fill with wax crayons all the parts that you dont want the fluid pass
?. [Channel Formation]
Melt the wax, we use a regular oven at 100 °C for 140s. Remove it from the heat and let the wax solidify. It must cross the paper.
?. [Acrylic paper sandwich]
Put the paper in between the acrylic sheets. You can put some tape to make it more impermable.
?. [Device Assemble]
Put the acrylic paper sandwich in the lid of the hardware.
?. [Device Assemble]
Introduce the paper ends in the Source and close the device. You can use a tip box as pint of support.
?. [Device Assemble]
Insert the paper acrylic sandwich in the clip to keep pressure in it and put the filter. Now you are ready to do experiments.
?. [Experiment workflow]
Put your favorite bacteria direct in the channels or you can use paper discs previusly cultured with bacteria.
?. [Experiment workflow]
Set your experiment. In this case we introduce the device in an incubator and get results with FluoPi a Low-Cost and Open-Acces transiluminator. Fabricate your own FluoPi
?. [Experiment workflow]
Get Results.
TITLE: Bogus Data Acquisition Protocol IV
AUTHORS: Abby   Moore

[DESCRIPTION]
The purpose of this protocol is to demo protocol development.

[BEFORE_START]
Before you start .....

[GUIDELINES]
Guidelines ....        

[STEPS]
1. In this step, I'll refer to the software that you need to use. I used the Software component to do this.


 

2. In this step, I'll refer to an image using embed code from Box.


 

3. In this step, I need to direct to to execute one of the following protocols depending on your context. Then you need to return to this protocol and continue with the next step. The two protocol options are hyperlinked below.


Bogus data acquisition protocol II
Bogus Data Acquisition Protocol III

4. Run the command displayed below, which is display with the command component.
TITLE: Knee Cartilage Grading Protocol 
AUTHORS: molmer  , Martin  Lotz

[DESCRIPTION]
The attached document shows the Gross Morphological Assessment Sheet we use to score and grade knee cartilage.

[STEPS]
SECTION: Gross Morphological Assessment Sheet
1. Macroscopic scores (1-4) are assigned to cartilage surfaces during tissue harvesting and recorded on the Gross Morphological Assessment Sheet (See Description). These scores use a modified Outerbridge classification grading system.

Score of 1 = normal, intact surface
Score of 2 = minimum fibrillation
Score of 3 = overt fibrillation, fissuring
Score of 4 = erosion
SECTION: Gross Morphological Assessment Sheet
1.1. Each grid space on the assessment sheet is assigned a score, as seen above 1-4, for cartilage on the Patella, Trochlea, Medial Femoral Condyle, Lateral Femoral Condyle, Medial Tibial Plateau, and Lateral Tibial Plateau.
SECTION: Gross Morphological Assessment Sheet
2. The individual grid scores for each knee are added together ( left and right knees are added up separately). Each sum will range from 48 to 192.
SECTION: Assessing the Total Knee Cartilage Grade
3. The total score for each knee will determine the grade category with normal (Grades 0 or I) or OA (Grades 3 or 4).

Knee Cartilage Grades 0-IV:
Grade 0 = normal (total score of 48)
Grade I = minimal change (total score 49-72)
Grade II = mild change (total score 73-96)
Grade III = moderate change (total score 97-120)
Grade IV = severe change (total score 121+)
TITLE: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer
AUTHORS: J Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Proteolytic peptide measurement using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer for peptide/protein identification and quantification.




[STEPS]
1. Dilute the peptide sample with solvent A (2% ACN, 0.1% FA) to the desired concentration. Transfer the samples to an autosampler vial and add iRT peptides to the sample according to the manufacturer’s instructions.
2. Inject the sample onto a Dionex UltiMate 3000 liquid chromatographic system coupled to an Orbitrap Eclipse Tribrid mass spectrometer and load the peptides on a C18 trap column over 10 min at 5 µL/min with 100% solvent A.
3. Elute the peptides with a C18 analytical column heated at 35°C and at a flow rate of 300 nL/min over 215 min using the following gradient of solvent B:
3.1. 2% for 10 min
3.2. From 2% to 20% in 125 min
3.3. From 20% to 32% in 40 min
3.4. From 32% to 80% in 1 min
3.5. 80% for 9 min
3.6. From 80% to 2% in 1 min
3.7. 2% for 29 min.
4. Acquire the data in data-independent acquisition (DIA) mode using the settings described in Table 1. One cycle consists of one full MS scan collected in the Orbitrap analyzer followed by DIA-MS/MS scans collected in the Orbitrap analyzer based on an isolation scheme of 26 variable windows covering the 350 - 1,650 m/z range with an overlap of 1 m/z (Table 2).
TITLE: Immunohistochemistry for brain sections
AUTHORS: Santiago Unda, Roberta Marongiu, Michael G. Kaplitt

[DESCRIPTION]
Protocol for processing 30um mouse brain sections for immunolabeling.

[STEPS]
SECTION: Tissue Preparation
1. Sections at cryostat:
Thickness 30 µm
Sections picked up with a wet brush (not too wet) and moved to the TBS-T wells (usually serial sections). Store section in anti-freeze solution in -20 °C if not immediately staining.

SECTION: Day 1: Primary Antibody Incubation
3. Washes: 3-step wash, 10 min each, on a shaker, in TBS-T. (Between washes, just move
the inserts from one tray to another.)
SECTION: Day 2: Secondary Antibody Incubation
7. Washes: a 3-step wash, 10 mineach, in TBS-T.
SECTION: Tissue Preparation
2. Transfer the sections into the net-wells.
SECTION: Day 1: Primary Antibody Incubation
4. Blocking step: add blocking solution into a 6-well plate.
Transfer the sections with a clean brush from the net-inserts into the 6-well plate.
Incubate on the shaker for 60 min at Room temperature.
SECTION: Day 1: Primary Antibody Incubation
5. Primary antibody: Transfer the sections into a 6-well plate containing the primary antibody solution (1 ml per well).
SECTION: Day 1: Primary Antibody Incubation
6. Incubation: on a shaker in the cold room 60 min.
SECTION: Day 2: Secondary Antibody Incubation
13. Mounting: Mount sections and let them dry for 10 min atRoom temperature.
SECTION: Day 2: Secondary Antibody Incubation
12. Washes: a 2-step wash, on a shaker, atRoom temperature, 10 min each.
SECTION: Day 2: Secondary Antibody Incubation
11. DAPI: incubate with DAPI (1:10,000) shaking for10 min at Room temperature.
SECTION: Day 2: Secondary Antibody Incubation
10. Washes: 3-4 times, for 10 min, in TBS-T.
SECTION: Day 2: Secondary Antibody Incubation
9. Incubation: on a shaker, at Room temperature, for 1-2 hours.
SECTION: Day 2: Secondary Antibody Incubation
8. Secondary antibody: Transfer the sections into a 12-well plate containing the secondary antibody. The secondary antibody will be incubated atRoom temperature. 
SECTION: Day 2: Secondary Antibody Incubation
14. Coverslip: Let the slides dry at least 10 minat Room temperature.
TITLE: Nuclear RNA purification
AUTHORS: Michael Tellier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for purifying nuclear RNA for qRT-PCR or next generation sequencing analysis.</div></div>

[STEPS]
?. [Day 1]
?. [Day 2]
RNA extractionWash the cells twice with ice-cold PBS.Scrap the cells in 1.2 ml of ice-cold PBS.Centrifuge at 1,000 rpm for 5 minutes at 4°C.Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B (10 mM Tris-HCl pH 8, 140 mM NaCl, 1.5 mM MgCl2, 0.5 % NP-40).Centrifuge at 1,000 g for 3 minutes at 4°C.Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B and transfer to an ice-cold 14 ml round-bottom tube.Add drop by drop 100 μl of the Detergent Stock Solution (3.3 % (w/v) sodium deoxycholate, 6.6 % (v/v) Tween 40) under slow vortexing.Transfer to a fresh ice-cold 1.5 ml tube. Centrifuge at 1,000 g for 3 minutes at 4°C.Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B.Centrifuge at 1,000 g for 3 minutes at 4°C.Resuspend the pellet in 1 ml of TRIzol using a 21-gauge syringe and incubated 5 minutes at room temperature.Add 200 μl of chloroform and vortex the sample vigorously for 15 seconds.Centrifuge at 12,000 g for 15 minutes at 4°C.Transfer the aqueous fraction to a new tube containing 580 μl of isopropanol.Incubate 10 minutes at room temperature.Centrifuge at 12,000 g for 10 minutes at 4°C.Remove most of the supernatant with a 1 ml pipette.Centrifuge at 12,000 g for one minute at 4°C.Remove the remaining liquid with a 10 or 20 μl tip.Resuspend the pellet in 87 μl of RNase-free water, 10 μl of 10 × DNase buffer, 2 μl of DNase I, and 1 μl of RNase OUT.Incubate for 30 minutes at 32°C.Add 100 μl of acid-phenol:chloroform pH 4.2.Vortex 10 seconds, then centrifuge 5 minutes at 13,000 rpm at room temperature. Transfer the upper phase to a new tube and add 100 μl of acid-phenol:chloroform pH 4.2.Vortex 10 seconds, then centrifuge 5 minutes at 13,000 rpm at room temperature. Transfer the upper phase to a new tube and add 250 μl of 100% ethanol, 10 μl of NaOAc, and 1 μl of Glycoblue (or 1 μl of glycogen 10 mg/ml).Invert the tube several times and incubate at -20°C for at least two hours (or overnight).Centrifuge for 20 minutes at 13,000 rpm at 4°C.Remove the supernatant and centrifuge for two minutes at 13,000 rpm at 4°C.Remove the last drops, air dry for 1-2 minutes, and resuspend in 20 ul of RNase-free water.Determine the concentration and the 260/280 and 260/230 ratios using a NanoDrop or another system.Perform the cDNA reaction for qRT-PCR or NGS.