eamag/protocols-with-steps huggingface dataset:
TITLE: SPARC_Duke_Grill_OT2-OD025340_VagusNerve_IHC_TH
AUTHORS: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes immunohistochemistry with anti-tyrosine hydroxylase, as it has been applied to cervical and abdominal vagus nerve samples from rats, pigs, and humans.</div></div>

[STEPS]
?. [Immunohistochemistry]
Bake slides with sections of paraffin-embedded vagus nerve overnight at 50oC and then cool overnight.
?. [Immunohistochemistry]
Deparaffinize the slides and hydrate them to distilled water: xylene (2x 6 min), 100% ethanol (5 min), 95% ethanol (4 min), 70% ethanol (3 min), deionized water (2x 1 min).
?. [Immunohistochemistry]
Perform heat-induced epitope retrieval (HIER) at 120oC for 30 s followed by 90oC for 10 s, using a buffer with pH 6.0 (Thermo, TA-135-HBL).
?. [Immunohistochemistry]
Cool for 20 min at room temperature.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Block with 3% H2O2 diluted in deionized water for 10 min.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Block using DAKO Protein Block (X0909) for 10 min at room temperature.
?. [Immunohistochemistry]
Apply the primary antibody (rabbit anti-tyrosine hydroxylase, Abcam, ab112) diluted in Thermo Antibody Diluent to a concentration of 1:250, and incubate overnight at 4oC.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply the secondary antibody (biotinylated SP-conjugated Affinipure goat anti-rabbit IgG (H+L), Jackson, 111-065-144) diluted in Thermo Antibody Diluent to a concentration of 1:500, and incubate for 1 hour at room temperature.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply ABC Elite (Vector, PK-6100) at a concentration of 1:50 for 30 min at room temperature.
?. [Immunohistochemistry]
Rinse in Tris Tween buffer (2x 2 min).
?. [Immunohistochemistry]
Rinse in Tris buffer (1x 2 min).
?. [Immunohistochemistry]
Apply DAB chromogen (Thermo, TA-125-QHDX) for 3 min at room temperature.
?. [Immunohistochemistry]
Rinse in deionized water (2x 2 min).
?. [Immunohistochemistry]
Counterstain using hematoxylin.
?. [Immunohistochemistry]
Dehydrate, clear, and coverslip using DPX mountant.
?. [Microscopy]
Each sample was imaged at 20x using a Nikon Ti2 microscope with a DS-Ri2 color CMOS camera (Nikon Instruments Inc.). We selected the best of four slices for each sample based on the quality of the slice (no tearing or fraying).
TITLE: Simple Banana Bread Recipe
AUTHORS: Anita Bröllochs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a simple 6-ingredients vegan banana bread recipe. </div></div>

[STEPS]
?. [Pre-heating oven]
Preheat the oven to .
350 °F
?. [Making the batter]
Add  and  into a medium-sized bowl.
[flour]
[baking powder]
?. [Making the batter]
Mix the dry ingredients well.
?. [Making the batter]
Mash the bananas with a fork.
?. [Making the batter]
Transfer the mashed banana into another medium-sized bowl.
?. [Making the batter]
Add , , and  to the mashed banana.
[oil]
[maple syrup]
[vanilla extract]
?. [Making the batter]
Mix all the wet ingredients well.
?. [Making the batter]
Add the wet ingredients to the dry ingredients and mix everything.
Be sure to not overmix as overmixing will cause the banana bread to be less fluffy.
?. [Making the batter]
Grease a baking pan with a neutral oil of your choice.
?. [Making the batter]
Fill the dough into the baking pan.
?. [Baking ]
Bake the banana bread at  for .
350 °C
?. [Baking ]
Let the banana bread cool down a bit before serving.
?. [Making the batter]
aDecorate the banana bread with pecans.
TITLE: Protein interaction modeling
AUTHORS: Shiyi Wang

[DESCRIPTION]
Protein interaction modeling using Alphafold 2.0 Multimer

[STEPS]
1. **Structure Prediction in AlphaFold 2.0 Multimer** - Use full-length mouse Ezrin (UniProt ID: Q4KML7) and Atg7 (UniProt ID: Q9D906) for structure prediction. - Predict the structure of Ezrin in the open conformation: - Independently predict Ezrin N- and C-termini. - Thread them onto an open ERM hinge structure using PyMOL2 molecular visualization software.
2. **Modeling Ezrin:Atg7 Interaction** - Predict the structures of Ezrin and the Atg7 homodimer using AlphaFold 2.0 Multimer. - Dock the predicted structures to model the Ezrin:Atg7 interaction and associated conformational changes.
3. **Assess Conformational Changes** - Use CABS-flex 2.0 (REF: https://doi.org/10.1093/nar/gky356) and PyMOL2 to assess conformational changes in the Ezrin:Atg7 protein complex.
4. **Select High-Confidence Structures** - For all predicted structure models, select the highest-confidence structures based on the predicted local distance difference test (pLDDT).
5. **Energy Minimization** - Subject the highest-confidence structures to energy minimization via AMBER relaxation.
6. **Import and Visualize Minimized Structures** - Import the minimized structures into PyMOL2. - Use PyMOL2 to produce representative models of the structures.
TITLE: BASIC PROTOCOL 4: Pan-genome Copy Number Variant Calling
AUTHORS: miriam.goldman  , chunyu.zhao  

[DESCRIPTION]
This protocol describes the CNV module of MIDAS2, which takes as input metagenomic sequencing reads from a set of samples and generates files with CNV genotypes for each sample for all detected species. There are two steps for population CNV calling: (1) single-sample quantification of copy number for each gene in the pangenome of each species with the midas2 run_genes command and (2) population CNV calling with the midas2 merge_genes command. Basic Protocols 1 (Species) and 2 (MIDASDB) should be run before this protocol.

[STEPS]
1. Perform species prescreening as described in Basic Protocol 1.
 

2. Download MIDASDB as described in Basic Protocol 2.
 

3. Execute the run_genes command for each sample
 

 

 

4. Prepare sample manifest file for merging purpose. We can use the same list_of_samples.tsv generated by step 6 in Basic Protocol 1.
 

5. Upon the completion of run_genes for all the samples listed in the list_of_samples.tsv, MIDAS2 merges the CNV profiles across samples with the merge_genes command.
 

6. Population pangenome CNV analysis has finished successfully when all the following output files are created under the directory midas2_output/merge/genes/ without any error message
TITLE: Sample vitrification and cryo-EM data acquisition
AUTHORS: Minghao Chen

[DESCRIPTION]
Sample vitrification and cryo-EM data acquisition

[STEPS]
SECTION: Sample vitrification
1. Glow-discharge the grids at 25 mA for 30 sec with PELCO easiGlow system (Ted Pella) 
SECTION: Sample vitrification
2. Apply 3 μl of protein solution to the grid:
QUANTIFOIL R1.2/1.3 mesh 300 (Electron Microscopy Sciences), or
QUANTIFOIL R2/1 mesh 300 (Electron Microscopy Sciences) 
SECTION: Sample vitrification
3. Vitrify the sample with a Vitrobot cryo-plunger (Thermo Fisher Scientific)
SECTION: cryo-EM data acquisition at 300 kV Titan Krios microscope
5. Collect dataset at a magnification of 81,000x and a corresponding pixel size of 1.05 Å and a defocus range of -0.8 to -2.0 μm. Image stacks contain 50 frames with a total dose of 50 e/Å2. 





SECTION: cryo-EM data acquisition at 200 kV Talos Arctica microscope
6. Collect dataset at a magnification of 36,000x and a corresponding pixel size of 0.5575 Å and a defocus range of -0.8 to -2.0 μm in a super-resolution correlated-double sampling mode. Image stacks contain 50 frames with a total dose of 50 e/Å2. 
SECTION: Sample vitrification
4. (Optional) Add 0.05%(w/v) of n-Octyl-Beta-D- Glucopyranoside as a surfactant before vitrification.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Obesity can be induced by exposing mice to a western-type diet high in sugar and fat for 15 weeks. Control animals receive a standard fat diet for 15 weeks.</p>

[GUIDELINES]
<p><span lang="EN-US" style="margin: 0px; line-height: 107%; font-family: 'Calibri',sans-serif; font-size: 11pt;">-HFD is stored at +4°C<br /> -Mouse needs to be out of cage, relaxed on a bench, a scalpel is used to cut of 1 mm of tail, the tail starts to bleed, glucose levels are measured on the second drop of blood.</span></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Proteolytic Peptides from Conditioned Media Desalting  with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges 
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Desalting of proteolytic peptides from conditioned media elution using C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges in preparation for downstream proteomic profiling. 

For desalting of proteolytic peptides from conditioned media Step 4 is modified as we typically reconstitute in an appropriate volume of 0.2% FA rather than by weight/volume due to the input material for digestion also being based on a proportion of the volume of concentrated conditioned media. 

[STEPS]
1. Centrifuge the samples at 1,850 x g for 5 minutes at room temperature to pellet insoluble material.
2. Desalt the samples using Oasis HLB solid-phase extraction cartridges placed on top of a vacuum manifold as follows:
2.1. Condition each cartridge two times with 800 µL of the condition buffer (50% ACN, 0.2% FA).
2.2. Equilibrate each cartridge three times with 800 µL of the equilibration buffer (0.2% FA).
2.3. Load the peptide samples.
2.4. Wash each cartridge three times with 800 µL of the wash buffer (0.2% FA). 
2.5. Elute peptides with 800 µL of the elution buffer (50% ACN, 0.2% FA) followed by a further 400 µL of the same elution buffer.
3. Vacuum dry the eluted peptide solution in a centrifugal vacuum concentrator.
4. Reconstitute the dried peptides in an appropriate volume of 0.2% FA and thoroughly mix the solution. 
5. Centrifuge at 12,000 x g at room temperature for 2 min, and store at -20°C.
TITLE: U Mass - Chronic drug delivery
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">A subcutaneous or intraperitoneal implantation of Alzet osmotic pump is used to chronically administer selected drug in mice. Chronic drug delivery may be used to examine intermediate to long-term effects of selected drug on obesity, insulin resistance, and metabolism.</div></div>

[STEPS]
?. Anesthetize mice with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight).
?. Shave hair at the incision site on the back.
?. Make an incision (~0.5 cm) using sterilized scalpel between the scapulae.
?. Subcutaneously insert an Alzet mouse osmotic pump containing drug or placebo.
?. Suture or close the incision site using sterilized staples.
?. Administer ketoprofen to minimize pain and house mice individually.
TITLE: Rab7 Phosphorylation reaction
AUTHORS: Dan Tudorica

[DESCRIPTION]
Phosphorylation of Rab7 at S72 using TBK1

[STEPS]
SECTION: Phosphorylation reaction
1. Mix phosphorylation reaction mixture. Combine 4 uM human Rab7 with 400 nM purified human strep-tagged TBK1 in a buffer consisting of 50 mM HEPES 7.5, 150 mM NaCl, 10 mM MgCl2, and 200 uM ATP. Incubate at room temperature for 3 hours.

SECTION: Phosphorylation reaction
2. Pass reaction mixture over a strep-tactin sepharose gravity column. Pass reaction mixture over resin x4 in order to remove all strep tagged TBK1 from solution. 
SECTION: Phosphorylation reaction
3. Buffer exchange via dilution and centrifugation or dialysis overnight in order to recover purified Rab7
SECTION: PhosTag gel
4. In order to assess degree of phosphorylation, prepare a 15 ug sample of phosphorylated Rab7 for PhosTag gel electrophoresis. Buffer exchange the protein into pure water via dilution and concentration with centrifugal concentrator.

As a positive control, prepare a sample of purified Casein, purchasable in bulk as a lyophilized solid. This protein is natively phosphorylated. Incubate with lambda phosphatase in order to produce its unphosphorylated form. Run the native casein as a positive control for the PhosTag gel, and the unphosphorylated form as a negative control. 
SECTION: PhosTag gel
5. Follow guidelines for Fujifilm Wako's "SuperSep" precast PhosTag gel. Add loading buffer to 1x, and run gel at ~120 V until the dye front reaches the bottom of the gel.
SECTION: PhosTag gel
6. Stain gel via Coomassie staining, and image.
TITLE: Protocol for Preparing Brain Samples for MUSIC
AUTHORS: Wenxin Zhao, Sheng Zhong

[DESCRIPTION]
Here states the detailed procedure to prepare brain samples for MUSIC study.

[STEPS]
SECTION: Tissue pulverization and crosslinking
1. Cut a portion of post-mortem human brain frontal cortex sample on dry ice with heavy razor blades, and collect 50 mg of the sample in a 1.5 mL LoBind tube. 
SECTION: Nuclei isolation
8. Use Chromium Nuclei Isolation kit (10X genomics, 1000494) to isolate nuclei from crosslinked cortex samples. 
SECTION: Nuclei isolation
9. Transfer 50 mg frozen tissue into pre-chilled sample dissociation tube. 
SECTION: Nuclei isolation
10. Add 400 μL of Lysis Buffer to Sample Dissociation Tube. Dissociate tissue with plastic pestle until homogeneous. 

 

SECTION: Nuclei isolation
12. Equally load the solution into two nuclei isolation column, and centrifuge the tubes at 16,000 x g for 20 sec at 4°C.
SECTION: Nuclei isolation
13. Vortex the flowthrough in the collection tube that contains nuclei for 10 sec at 3,200 rpm or max speed to resuspend nuclei. 
SECTION: Nuclei isolation
14. Centrifuge the collection tubes for 3 min at 500 x g at 4°C to pellet nuclei. Carefully discard the supernatant. 
SECTION: Tissue pulverization and crosslinking
2. Thaw the 50 mg of brain sample on ice, and chop the tissue into smaller pieces by pestle. Store the rest of the sample at -80°C.
SECTION: Tissue pulverization and crosslinking
3. Incubate the sample with 10 mL of 2 mM disuccinimidyl glutarate (DSG) in 1X PBS in a 15 mL LoBind tube at room temperature for 45 min with gentle rotation. 
SECTION: Tissue pulverization and crosslinking
4. Wash once with 10 mL of 1X PBS by centrifugation at 1,000 x g for 4 min.
SECTION: Tissue pulverization and crosslinking
5. Resuspend the sample in 15 mL of 1X PBS containing 3% formaldehyde, and incubate for 10 min with a gentle rotation.
SECTION: Tissue pulverization and crosslinking
6. Quench the crosslinking reaction by the addition of 5 mL of 1.25 M glycine followed by an incubation of 5 min with a rotation.
SECTION: Tissue pulverization and crosslinking
7. Centrifuge the sample at 1,000 x g for 4 min, and wash the sample twice with ice-cold 1X PBS containing 0.3% BSA (wt/vol).
SECTION: Nuclei isolation
11. Add 600 µL of lysis buffer into the tube, and mix 10 times by pipetting. Incubate on ice for 10 min.
SECTION: Nuclei isolation
15. Resuspend the nuclei in 500 µL of Debris Removal Buffer provided by the kit by pipetting 15 times. 

 

SECTION: Nuclei isolation
16. Centrifuge the nuclei at 700 x g for 10 min at 4°C. Carefully discard the supernatant. 
SECTION: Nuclei isolation
17. Resuspend the nuclei in 1 mL of Wash and Resuspension Buffer. 

 

SECTION: Nuclei isolation
18. Centrifuge the nuclei at 500 x g for 5 min at 4°C. Carefully discard the supernatant. 
SECTION: Nuclei isolation
19. Resuspend the nuclei again in 1 mL of Wash and Resuspension Buffer. 
SECTION: Nuclei isolation
20. Centrifuge the nuclei at 500 x g for 5 min at 4°C. Carefully discard the supernatant as much as possible. 
SECTION: Nuclei isolation
21. The nuclei are subjected to nuclei counting and the following procedures.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/CviJI-Purification-From-IL-3A-Virus-Infected-NC64A-er3bd8n" target="_blank">CviJI Purification From IL-3A Virus Infected NC64A Chlorella</a>.

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

[DESCRIPTION]
Preparation of electrocompetent cells 
Weinstock paper:
Matthew T Weinstock, Eric D Hesek,Christopher M Wilson, Daniel G Gibson
Vibrio natriegens as a fast-growing host for molecular biology
Nature Methods volume 13, pages 849–851 (2016)
 
 
To prepare the day before:


-LB I media  +V2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2 )
-electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7) ( sterile filtrated)
-over night culture of  V.n from the cryo-stock in LBI + v2 salts (37 °C ; at 200 r.p.m)
 
Preparing culture :
 
Depending on how many aliquds you want to have, incubate media with your overnight culture for a starting OD of 0.05.
The culture is grown at 37 °C in a baffled flask, shaking at 200 r.p.m. until an OD600 between 0.5 to 0.8 is reached.
*be careful when they reach an OD near 0.1, V. natriegensis very fast growing,  so start measuring in shorter time periods.
Prechill the electroporation buffer
 
Washing:
 
From here on try always to keep your culture on ice.
 
-The culture is then put on ice for 15 min. 
 ( the original Protocol sais to directly fill them into your prechilled centrifugatin  
  containments )
-The cells are pelleted at 3000x g. for 20 min at 4 °C.
-The supernatant is carefully decanted and the cell pellets are gently suspended in 10mL
 of chilled electroporation buffer.
-The suspensions are transferred to a chilled50mL falcon tube and the tube is filled top
 with additional chilled electroporation buffer (50mL) and inverted several times. 
-The cells are centrifuged down at 3000x g  for 15 min at 4 °C. 
 
-The wash is repeated two times for a total of three washes.
 
Aliquotation:
 
After the final wash, the supernant is carefully decanted the cells are gently resuspended in residual electroporation buffer. 
Measure the OD in a  1/20 dilution against electroporation buffer.
 
The volume is adjusted with additional electroporation buffer to bring the final OD600 to 16.
  
The Cells are aliquoted ( 80µL ) into chilled 1.5µL centrifugation tubes, directly frozen in liquid nitrogen and stored at −80 °C until use.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Dyes are colored organic compounds that can dye fibers or other substrates to a certain color. They can be divided into the following categories: Acid Dyes, Azoic Dyes, Basic Dyes, Direct Dyes, Cationic Dyes, Disperse Dyes, Neutral Dyes, Pigments, Reactive Dyes, Solvent Dyes and so on. 

[STEPS]
TITLE: Digging Test
AUTHORS: sdwalto, Jeffrey Kordower, Bryan_Killinger

[DESCRIPTION]
Digging test optimized for mice. This is a test to assess olfaction impairment. The more olfactory impairment or deficits, the longer it will take to find the sweetened treat.  

[GUIDELINES]
Note: If the treat was found within 5 mins, the animal was allowed to eat it. 

[STEPS]
1. Fast mice overnight (Do not exceed 24 hours). 
2. Prepare a clean mouse cage filled with 3 cm of bedding. 
3. Prepare an appetitive stimulus of sweetened cereal. Cinnamon Toast Crunch was used here. 
4. Bury cereal piece 0.5 cm below the bedding along the perimeter of the cage. 
5. Monitor the mouse for 5 mins or until they find the treat. Time was recorded until the treat was found. If treat was not found by the end of 5 mins, mouse does not get the treat. 
6. Replace the bedding and repeat for each mouse.
TITLE: Production of Crude AAV Virus Extract
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to produce crude preps of AAV of any serotype.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Numbers RF1MH114126 and RF1MH121274. 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: A study of the layout planning of plant facility based on the timed Petri net and systematic layout planning
AUTHORS: Hanwen Liu, Hanwen Liu, Xiaobing Liu, Lin Lin, Yuqing Xu, Sardar Mn Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A study of the layout planning of plant facility based on the timed Petri net and systematic layout planning</div><div class = "text-block">The purpose of this research is to solve the problems of unreasonable layout of the production plant, disorder of the logistics process, and unbalanced production linein discrete manufacturing plants. </div></div>

[STEPS]
TITLE: Cleavage assay
AUTHORS: Yuichiroh  Ikagawa

[DESCRIPTION]
Cleavage assay to validate for Cas12a activity 

[STEPS]
1. Thawed crRNA solution on ice.
2. Add the following solution to the tube and gently mix by pipetting.

 
If the crRNA or Cas12a solution was not added to the reaction as a negative control, the reaction buffer for it was added and the total volume was adjusted to 20 μl.
5. Incubate the mixture at 37°C overnight.
6. Check for cleavage by1.5% agarose gel electrophoresis. 
3. Incubate for 10 minutes.
4. Add 4μl of U94 solution to each tube.
TITLE: HTTM : Illumina library preparation
AUTHORS: Antoine Champie

[DESCRIPTION]
Part of the HTTM protocol dedicated to the preparation of Illumina sequencing libraries.

[BEFORE_START]
All steps and master mixes need to be kept on ice  as much as possible. Thermocyclers need to be cooled at 4C before inserting sample plate.  

[STEPS]
SECTION: Libraries
1. Transfer 2.5µl of DNA from the DNA extraction plate to a new PCR plate. 
SECTION: Libraries
2. Prepare a fragmentation master mix with : 

 

SECTION: Libraries
3. Add 1 µL of the fragmentation master mix to each well. 

SECTION: Libraries
4. Incubate in a thermocycler with the following protocol : 
15 minat 37 °C
30 minat 65 °C




SECTION: Libraries
5. Add 1 µLof 4µM Nextera (NxT) adaptors to each well.

SECTION: Libraries
6. Prepare a ligation master mix with : 
 

SECTION: Libraries
7. Add 3.5 µLof ligation master mix to each well.

SECTION: Libraries
8. Incubate in a thermocycler with the following protocol : 
30 minat 20 °C
10 minat 65 °C

SECTION: Libraries
9. Prepare a PCR master mix with : 

 

SECTION: Libraries
10. Add 192 µLof PCR master mix to each well.

SECTION: Libraries
11. Split the PCR reaction into 4 different plates (50µl per plate). 
SECTION: Libraries
12. Incubate each plate in a thermocycler with the following cycles : 
30 sat 98 °C
15 sat 98 °C 
30 sat 72 °C
Repeat from step 2 for 20~25 cycles*
2 min  72 °C






SECTION: Libraries
13. Pool the 4 PCR replicates together in a.
SECTION: Libraries
14. Transfer2 µLof DNA from the pool plate to a new PCR plate.

SECTION: Libraries
15. Add 2 µLof each barcoding primer to the DNA : 
- Nxt_i5_barcoding
- Nxt_i7_barcoding

SECTION: Libraries
16. Prepare a PCR master mix with : 

 
 
 

 
 
SECTION: Libraries
17. Add 44 µL of the PCR master mix to each well of the plate. 

SECTION: Libraries
18. Incubate in a thermocycler with the following protocol : 
30 s at 98 °C
15 s at 98 °C
1 min at 72 °C (no anneal step)
Repeat from step 2 for 5 cycles
2 min at 72 °C






SECTION: Libraries
19. Pool together 2 µL of each sample.

SECTION: Libraries
20. Purify with SPRI beads using a 0.8 ratio. Resuspend with50 µL of molecular grade water.

SECTION: Libraries
21. Proceed with QC and sequencing.