eamag/protocols-with-steps huggingface dataset:
TITLE: ELISA for quantification of IL-6 in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukin-10 is a pleiotropic cytokine playing a critical role as a regulator of myeloid and lymphoid cell function. Due to the ability of IL-10 to blocking cytokine synthesis and many accessory cell functions of antigen-presenting cells such as macrophages this cytokine is a potent suppressor of the effector functions of macrophages, T-cells and NK cells. IL-10 also participates in regulating proliferation and differentiation of B-lymphocytes, mast cells and thymocytes [1]. </div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Ouyang W, O'Garra A. IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation.</span><span style = "font-style:italic;">Immunity</span><span>. 2019;50(4):871-891. doi:10.1016/j.immuni.2019.03.020</span></div></div>

[STEPS]
?. An anti-human IL-6 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-6 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wasth o remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-6 antibody is added.
?. The microplate is rewashed with PBS-Tween buffer.
?. One hundred µl of streptavidin-HRP is added and binds to the biotin-conjugated anti-human IL-6 antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 50 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-6 present in the sample or standard.
?. The reaction is terminated by addition of 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-6 standard dilutions and the human IL-6 sample concentration determined.
?. For better results place the microplate on a shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The aim of this study was to systematically review and analyze the published preclinical studies of MSC administration in the treatment of animal models of PQ poisoning to provide a basis for cell therapy of PQ poisoning.The electronic databases PubMed and CBMdisc were searched in this systematic review. The characteristics of MSC treatment of animal models of PQ poisoning were summarized. After quality assessment, relevant results were used to evaluate the effects of MSC transplantation. Publication bias was assessed.</p>

[GUIDELINES]
<p>This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) criteria.</p>

[STEPS]
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TITLE: Protein extraction, quantification, and western blot for Bodo saltans
AUTHORS: Ewa  Chrostek,   Mastaneh Ahrar,   Gregory Dd Hurst

[DESCRIPTION]
This protocol is used in our Laboratory in Liverpool to work with proteins from Bodo saltans and other kinetoplastids (eg. trypanosomes). 

[STEPS]
SECTION: Culture conditions
1. Bodo saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na2HPO4)1. Cultures were incubated at 22 °C in T25 tissue culture flasks containing 20 ml of media bacterized with Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700831). 
SECTION: Protein extraction and quantification
1.1. Centrifuge 20 ml of 1 week old culture at 1200 × g for 12 mins at 19 °C to pellet the cells.
SECTION: Protein extraction and quantification
1.2. Dissolve pellet in lysis buffer2: 150 mM NaCl, 1% Triton X-100, 50 mM TrisHCl (pH8), 5 mM EDTA. Supplement lysis buffer with cOmplete Protease Inhibitor Coctail (Roche) (1 tablet per 10 ml of solution) and Benzonase nuclease (Sigma) immediately before extraction to prevent protein degradation and to decrease viscosity of extract, respectively. 
SECTION: Protein extraction and quantification
1.3. Incubate extract on ice for 30 mins.
SECTION: Protein extraction and quantification
1.4. Centrifuge at 8000 × g to pellet debris.
SECTION: Protein extraction and quantification
1.5. Collect supernatant.
SECTION: Protein extraction and quantification
1.6. Quantify protein (eg. using the Pierce™ BCA Protein Assay Kit, (Thermo Fisher) according to the kit instructions).
SECTION: Western blot
2. For western blot, adjust protein concentrations so that they are equal between samples. 
SECTION: Western blot
2.1. 1. Separate protein extracts on polyacrylamide gels (eg. Bolt Bis-Tris Plus, Thermo Fisher).
SECTION: Western blot
2.2. 2. Transfer proteins to PVDF membrane (eg. using mini blot module (Thermo Fisher)). 
SECTION: Western blot
2.3. 3. Block the membrane for 30 mins at room temperature in 5% skimmed milk diluted in wash buffer (PBS with 0.1% Tween20).
SECTION: Western blot
2.4. 4. Incubate the membrane overnight at 4 °C with primary antibodies (1:100 times diluted in 5% milk in wash buffer). 
SECTION: Western blot
2.5. 5. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20). 
SECTION: Western blot
2.6. 6. Incubate the membrane for 1 hour at room temperature with Horseradish Peroxidase (HRP)-labelled secondary antibodies (1:5000 dilution in 5% milk in wash buffer). 
SECTION: Western blot
2.7. 7. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20). 
SECTION: Western blot
2.8. 8. Develop the signal with HRP substrate (eg. Immobilon western kit, Merk) and scan for chemiluminescent signal (eg. with ImageQuant LAS4000 or Bio Rad imaging system). 
SECTION: Western blot
2.9.   

SECTION: References
3. 1. Gomaa Fatma, Li ZuHong, Docampo Roberto, Girguis Peter, E. V. Bodo saltans culture protocol V.2. Protocols.io (2018). doi:10.17504/protocols.io.sh6eb9e
2. Newton, I. L. G., Savytskyy, O. & Sheehan, K. B. Wolbachia Utilize Host Actin for Efficient Maternal Transmission in Drosophila melanogaster. PLOS Pathog.11, e1004798 (2015).
TITLE: Neuropathy Phentoyping Protocols - Intra-Epidermal Fiber Density Determination of Rodent Foot Pad Biopsies
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span style = "font-weight:bold;">Phenotyping of Rodents for the Presence of Diabetic Neuropathy </span></div><div class = "text-block">In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bitq4kcn.jpg" /></div></div>

[STEPS]
?. Perfused mouse or rat: 1. Following perfusion, remove entire foot pad with a sharp razor blade. Begin by cutting skin between the plantar surface of the foot and the toes, then start at the heel to gently remove the skin and superficial fascia of the foot. Be careful to only handle the tissue by the heel. 2. Post-fix overnight in 4% paraformaldehyde. 3. Rinse in graded sucrose 5 – 20%, minimum 12 hours in each
?. Fresh mouse or rat: 1. Remove foot and fix overnight (12-18 hours) in 4% paraformaldehyde 2. Remove skin and process as described above. 3. Follow IHC protocol using rabbit anti-PGP9.5, 1:1000 followed by AlexaFluor 598, 1:200 (Molecular Probes). Apply coverslips with ProLong antifade kit (Molecular Probes)
?. Imaging: Three images per sample are collected on an Olympus FluoView 500 confocal microscope using a 60 X 1.2 water immersion objective at a resolution of 800 X 600 pixels. The optical section thickness is 0.5 um. Forty images per stack are flattened using MetaMorph (version 6.14) arithmetic option. Integrated morphometry analysis is used to exclude extraneous signals. The data are presented as the percent area of PGP9.5 positive fibers per area of epidermis.
TITLE: Whole genome amplification of dengue virus type 1 - 4
AUTHORS: Anna Nagy

[DESCRIPTION]
Dengue virus is one of the most often imported tropical viral infection in Hungary. For next-generation whole genome sequencing of dengue virus serotypes 1 - 4, a one-step reverse transcription PCR assay was developed. PCR amplicons can be used for further amplicon based sequencing protocols on Illumina MiSeq platform. The genome of the dengue virus is a single-stranded, positive-sense, capped RNA of approximately 10–11 kb in length. The whole genome amplification was carried out by five overlapping PCR amplicons, each one is approximately 2500 nucleotide in length. For ampification of the four serotypes, four different primer sets were designed by Geneious Prime (version 2021.2.2) primer design tool. 

[STEPS]
1. Nucleic acid extraction: 
Use Qiagen QIAamp Viral RNA Mini Kit (cat. no. 52904 or 52906) for nucleic acid extraction. Nucleic acid extraction should be done by following the manufacturer's instructions. The total volume of the extracted viral RNA is 60 µl.
2. One-Step Reverse transcription (RT) PCR Setup
2.1. Reagent name: Invitrogen™ SuperScript™ III One-Step RT-PCR System with Platinum™TaqHigh Fidelity DNA Polymerase (cat. no. 12574030 or 12574035)

Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and immediately start the RT–PCR program. Dengue virus whole genome can be amplified by 5 overlapping fragments for each sample. Detailed description of primer sets for the whole genome amplification of DENV serotypes 1 - 4 can be found in the Excel sheet (Primers_DENV_whole_genome_amplification) attached below.   
PCR setup:
Add the following to a 0.2–mL, nuclease-free, thin-walled PCR tube on ice:
 

For multiple reactions, you can prepare a master mix to minimize reagent loss and enable accurate pipetting.


2.2. Program the thermal cycler so that cDNA synthesis is followed immediately with PCR amplification automatically:
 
Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed. Place the reaction tubes in the preheated thermal cycler programmed as described above. 
3. Agarose gel electrophoresis: for visualization and interpretation of the results 1% agarose gel should be used.
TITLE: High Efficiency Transformation Protocol (C2987H)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the protocol for C2987H cells. If you are using the C2987I cells, please refer to this protocol.

[BEFORE_START]
For this protocol, perform steps 1-8 in the tube provided. 

[GUIDELINES]
Transformation Protocol Variables

Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.

Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.

Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.

Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

DNA Contaminants to Avoid
   Contaminant Removal Method   Detergents Ethanol precipitate   Phenol Extract with chloroform and ethanol precipitate   Ethanol or Isopropanol Dry pellet before resuspending   PEG* Column purify or phenol/chloroform extract and ethanol precipitate   DNA binding proteins (e.g. Ligase) Column purify or phenol/chloroform extract and ethanol precipitate   



[STEPS]
1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 min.
 

2. Add 1 µL-5 µL containing 1 pg-100 ng to the cell mixture.
3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 min. Do not mix.
 

5. Heat shock at exactly 42 °C for exactly 30 s. Do not mix.
 

6. Place on ice for 5 min. Do not mix.
7. Pipette 950 µL into the mixture.
8. Place at 37 °C for 60 min, shaking vigorously (250 rpm) or rotating.
 

9. Warm selection plates to 37 °C.
 

10. Mix the cells thoroughly by flicking the tube and inverting.
11. Perform several 10-fold serial dilutions in SOC.
12. Spread 50 µL-100 µL of each dilution onto a selection plate.
13. Incubate 60 min at 37 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To isolate epithelial cells for sorting from the mouse small intestine </p>

[BEFORE_START]
<p>Put PBS into fridge for use. Only use cold PBS. </p>

[GUIDELINES]
<p>Work quickly and on ice. Keep PBS on ice throughout protocol to maintain cold. Keep extra PBS at +4.</p>

[STEPS]
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TITLE: Immunofluorescence staining on larval and adult Drosophila gonads
AUTHORS: Samantha Goetting

[DESCRIPTION]
Immunofluorescence staining protocol for Drosophila gonads

[BEFORE_START]
All steps are done with gentle rotation.

[STEPS]
SECTION: Day 1
1. Dissect tissue in 1x PBS. Transfer to a 1.5 mL tube containing 1x PBS. If it is a quick dissection (<20 mins), no ice needed. If more time is needed, keep samples on ice.
SECTION: Day 1
2. Remove PBS and add fixative. Fix in 4% paraformaldehyde in 0.3% PBTx for 20 min RT with gentle rotation. 
        500 uL fixative = 125 uL of 16% paraformaldehyde + 375 uL 0.3% PBTx 
SECTION: Day 1
3. Aspirate the fixative and wash twice for 10 min in 1% PBTx. Not getting rid of fix will affect your immunostaining.
SECTION: Day 1
4. Aspirate the supernatant and block/permeabilize for at least 2 hours in 1% PBTx + 5% normal serum, or overnight at 4°C.
        1 mL block solution = 50 uL NGS + 950 uL 1% PBTx
SECTION: Day 1
5. Primary antibodies are diluted in 0.3% PBTx + 5% normal serum and incubate for 1 hour at RT or overnight at 4°C. Overnight will give better staining. 
        Primary antibody mix in 0.3% PBTx + 15 uL NGS (300 uL total)
SECTION: Day 2
6. Remove the primary antibody mix and wash in 0.3% PBTx three times for 20 min at RT. 
SECTION: Day 2
7. Wash in 0.3% PBTx + 5% normal serum twice for 30 min at RT.
        1 mL wash solution = 50 uL NGS + 950 uL 0.3% PBTx
SECTION: Day 2
8. Secondary antibodies are diluted in 0.3% PBTx + 5% normal serum and incubated for ~2 hours at RT or overnight at 4°C. Keep tubes covered from light. 
SECTION: Day 2
9. Aspirate the supernatant and add 500 uL DAPI to each tube. Incubate for 10 min at RT. Keep tubes covered from light.
SECTION: Day 2
10. Aspirate the supernatant and wash in 0.3% PBTx three times for 20 min at RT.
SECTION: Day 2
11. Aspirate the supernatant and wash in PBS for 10 min at RT.
SECTION: Day 2
12. Store in PBS at 4°C or proceed with mounting. Keep tubes covered from light.
TITLE: Acid nucleic extraction from rice dried leaves 
AUTHORS: Martine Bangratz, Charlotte Tollenaere

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Acid nucleic extraction of rice dried leaves by a CTAB method adapted from Li et al 2008.</div><div class = "text-block"> Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods. 2008;154(1):48-55. doi: 10.1016/j.jviromet.2008.09.008.</div></div>

[STEPS]
?. Grind the dried leaves with the Qiagen TissueLyser II until obtaining a fine powder
?. Add 1 ml  CTAB extraction buffer (see 'before start' for the buffer content) and homogenize by vortexing.
?. Incubate at 65°C for 30 min. Periodically, mix gently the tubes during the incubation.
65 °C
Centrifuge: 10000 34, 10 min, 4 10
?. Transfer the supernatant (650µl) to a 1.5 ml microcentrifuge tube.Add equal volume of chloroform/isoamyl alcohol (24:1)
?. After shaking,
Centrifuge: 15000 34, 10 min, 4 10
?. Transfer the supernatant (500µl)  in a new 1.5 ml tube containing 350 µl of isopropanol (pre-chilled at -20°C).Mix gently
-20 °C
Centrifuge: 15000 34, 10 min, 4 10
?. Discard the supernatant . Wash the pellet with 70% ethanol.
Centrifuge: 15000 34, 5 min
?. Remove the  ethanol and air-dried the pellet.
?. Dissolve the pellet in 50 µl sterile water or TE buffer  and conserve the DNA at -20°C.
TITLE: Enzyme linked immunosorbent assay (ELISA) for determining the serum concentration of IL-17 in humans.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukin17A is a pro-inflammatory cytokine. This protein is produced by a group of T helper cell known as T- helper17 lymphocytes in response to their stimulation with IL-23. After interaction with its receptor, IL-17 activates signalling pathways that stimulate the production of chemokines [1 ].</div><div class = "text-block">Reference</div><div class = "text-block">1. Cho ML, Kang JW, Moon YM, Nam HJ, Jhun JY, Heo SB, Jin HT, Min SY, Ju JH, Park KS, Cho YG, Yoon CH, Park SH, Sung YC, Kim HY (May 2006). Journal of Immunology. 176 (9): 5652–61. </div></div>

[STEPS]
?. Ninety-six well ELISA plates are coated with monoclonal anti-human antibodies to interleukin-17( IL-17).
?. Patient serum samples are added to the plates.
?. The plate is incubate x 1.30 hour at RT.
?. The plate is washed 4 times with PBS-tween 20 buffer.
?. The wells are incubated with a biotin conjugated anti-human IL-17 for 1.30 hour at RT.
?. The plates are washed again as above.
?. To the plate a peroxidase-labeled streptavidin conjugate is added and incubated for 1 hour at RT.
?. After a further washing procedure a substrate solution reactive is added and allowed to produced a colored reaction in positive controls.
?. The level of IL-17 in the sample is proportional to the colored product developed.
?. The addition of acid stopped the reaction.
?. The absorbance is measured at 450 nm.
?. The IL-17 concentration can be calculated by generating an standard curve.
TITLE: Determine the Size of Sonicated Samples and the DNA Concentration
AUTHORS: Vasso Makrantoni, Daniel Robertson, Adele L. Marston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.</div></div>

[STEPS]
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
To a  add  containing .
[Input sample]
[TE buffer]
[NaCl]
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
Decross-link at .
65 °C
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
Add  and incubate at  for .
[RNase A (10 mg/ml)]
37 °C
RNase treatment is important as high levels of RNA will interfere with DNA purification when using commercially available PCR purification kits. DNA yield can be markedly reduced as the columns become saturated.
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
Add  and incubate at  for .
[Proteinase K (10 mg/ml)]
65 °C
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
Purify DNA using a PCR purification kit. Run purified DNA on a 2% agarose gel with a 100 bp DNA ladder marker to determine fragment size. Ideally sonication should yield an enrichment of fragments between 200 and 400 bp (Fig. 2a).
?. [Determine the Size of Sonicated Samples and the DNA Concentration]
Measure DNA concentration using a Qubit HS assay kit.
TITLE: Non-radioactive phosphorylation with T4 PNK 3&#39; phosphatase minus (M0236)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Protocol for non-radioactive phosphorylation with T4 PNK 3' phosphatase minus.

[STEPS]
1. Set up the following reaction in a microcentrifuge tube on ice:
 

 


2. Incubate at 37 °C for 30 min.
3. Heat inactivate by incubating at 65 °C for 20 min.
TITLE: Nuclei Isolation with EZ Prep Kit
AUTHORS: Le Zhang

[DESCRIPTION]
This protocol details nuclei Isolation with EZ Prep Kit.

[STEPS]
1. Prepare nuclei suspension buffer by adding2 µLof RNase Inhibitor (40 U/μl) per mL of nuclei suspension buffer. Vortex briefly to mix.

2. Keeping everything  on ice, add 1 mL  EZ PREP buffer (Sigma, NUC101) and tissue piece to the small 2 mL glass tissue grinder. Grind 3 times just to mash the tissue at the bottom of the grinder, then homogenize 25 times with pestle A. Add an additional 1 mL EZ PREP buffer and homogenize 25 times with pestle B. Move slowly to avoid spilling buffer. 

3. Use a pipette to transfer to 15 mL conical tube. Add 2 mL EZ PREP buffer to the tissue homogenizer to rinse and pipette into conical tube. Pipette gently to mix.

4. Incubate on ice  for  5 min.

5. Centrifuge for 5 min  at 500 x g  at 4 °C.

6. Remove supernatant carefully with a pipette. Do not disturb the pallet and leave behind some buffer. Resuspend in 1 mL EZ PREP buffer, then add 3 mL EZ PREP buffer to wash.

7. Incubate 4 °C  for  5 min.
8. Centrifuge for 5 min  at 500 x g  at 4 °C.
9. Remove supernatant carefully with a pipette. Resuspend in 1 mL nuclei suspension buffer, then add 3 mL nuclei suspension buffer. Pipette to mix.
 


10. Centrifuge for 5 min  at 500 x g  and 4 °C.
11. Remove supernatant carefully with a pipette. Resuspend in 500 µL nuclei suspension buffer.



12.  In a new labeled Eppendorf tube, make a 1:1 dilution of the nuclei suspension and Trypan Blue and count nuclei with hemocytometer slide (C-Chip, DHCN015).
 



12.1. If there are visible clumps of nuclei when counting, re-pipette gently 20 times and then incubate on ice  for 5 min. Make new 1:1 dilution with Trypan Blue and then recount and check for clumps of nuclei.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>This protocol is used to assess the sgRNA-to-barcode linkage in Mosaic-seq. The 3kb fragment can be amplified from either plasmids or genomic DNA extract from cells after lentivirual infection. The fragments are then circularized and used to amplify a smaller amplicon (about 270bp) which is compatible with Illumina sequencing.</p>

[STEPS]
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