TITLE: Preparation of PCR amplicons from edited cells for deep sequencing
AUTHORS: Mark DeWitt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to extract and amplify DNA from genome-edited cells, and sequence the resulting amplicons using Illumina MiSeq NGS.</div></div>

[STEPS]
?. [Prepare first PCR (genomic DNA PCR)]
Prepare the following Master Mix using the initial PCR primers (annealing outside the HDR template region, see guidelines for design requirements), and the PrimeStar GXL polymerase kit from Clontech. Include a master mix-only control.Master mix (per reaction):     0.15 µL 100 µM forward primer     0.15 µL 100 µM reverse primer     4 µL Takara dNTP solution (2.5 mM each, or 1 µL 10 mM each)     1 µL GXL polymerase     30.7 µL H20     10 µL 5X BufferConsider making 10-15% extra master mix to account for pipette error.
?. [Extract genomic DNA from edited cells using QuickExtract solution]
Resuspend cell pellet to ≥2,500 cells/µL in QuickExtract solution. Vortex or pipette-mix to resuspend thoroughly.
?. [Prepare first PCR (genomic DNA PCR)]
?. [Extract genomic DNA from edited cells using QuickExtract solution]
Place on 68ºC heat block or thermocycler. Incubate for 30 minutes.
?. [Extract genomic DNA from edited cells using QuickExtract solution]
Vortex or pipette-mix for 5 seconds.
?. [Extract genomic DNA from edited cells using QuickExtract solution]
Place on 98ºC heat block or thermocycler. Incubate for 15 minutes.
?. [Prepare first PCR (genomic DNA PCR)]
Mix 4 µL of genomic DNA extract with 46 µL of Master mix in a 96-well PCR plate. Add 46 µL master mix to an empty well as a master mix only control. Mix thoroughly with pipette. Seal plate with tape.
?. Purify 50 µL PCR with 1.8X SPRI beads, eluting in 30 µL water.1. Add 90 µL SPRI beads to each 50 µL PCR (1.8X ratio), and mix thoroughly.
?. [Purify first genomic PCR using SPRI beads]
2. Place on magnetic stand, wait for solution to clear (~1-2 minutes).
?. [Purify first genomic PCR using SPRI beads]
3. Remove clear supernatant.
?. [Purify first genomic PCR using SPRI beads]
4. Wash 2x with 80% Ethanol in water: Add 200 µL ethanol solution, remove, add 200 µL ethanol solution, remove.
?. [Purify first genomic PCR using SPRI beads]
5. Remove residual ethanol with a P20 pipette. Remove plate from magnetic stand.
?. 6. Allow sample to visibly dry, 3-5 minutes. Do not over-dry the samples as this can affect the yield.
?. [Purify first genomic PCR using SPRI beads]
7. Resuspend beads in 30 µL PCR-grade water. Place on magnetic stand.
?. 8. Allow solution to clear. Remove 27 µL of clear supernatant (contains DNA) and place in a new 96 well plate.
?. [QC the first PCR]
9. Measure concentration of first PCR using Nanodrop (A260) or Qubit Spectrophotometer.For qubit, use 2 µL Purified PCR per sample, and the 'DNA HS kit'. Follow manufacturer's instructions.
NOTE: The qubit assay can be readily adapted to use with a plate reader, for higher throughput.
?. [QC the first PCR]
2. Confirm proper amplification by running a 2% agarose gel with 5 µL of purified product. Alternatively, you can run the gel on the PCR, before purification.The first PCR product is often impure, especially when amplifying more complex samples (e.g. mouse samples). Confirm presence of the desired band before proceeding with the second PCR.
?. [Prepare the second PCR from 50 ng of product from the first PCR]
Place 50 ng of first PCR DNA into a second 100 µL GXL Primestar master mix with the second PCR primers. This produces an amplicon short enough to be sequenced on a MiSeq. Neither primer can anneal to the HDR template. See attached Word document for more design instructions.0.3 µL 100 µM forward primer 20.3 µL 100 µM reverse primer 28 µL Takara dNTP solution (2.5 mM each, or 1 µL 10 mM each)2 µL GXL polymeraseXX µL H20 (final volume: 100 µL)20 µL 5X BufferXX µL PCR1 product (50 ng)
?. [Prepare the second PCR from 50 ng of product from the first PCR]
Cycle using the following parameters:7 cycles of:            98ºC 10 sec            60ºC 15 sec            68ºC 30 sec
?. 
?. [Purify the Second PCR using SPRI beads]
2. Place PCR plate on magentic stand, allow solution to clear (2-3 minutes).
?. [Purify the Second PCR using SPRI beads]
3. Remove clear supernatant.
?. [Purify the Second PCR using SPRI beads]
4. Add 180 µL of 80% ethanol to wash. Remove, and repeat for a total of 2 washes.
?. 5. Remove residual ethanol with a P20 pipette.
?. [Purify the Second PCR using SPRI beads]
6. Remove plate from magnetic stand, let pellet air dry for 3-5 minutes. Do not overdry.
?. [Purify the Second PCR using SPRI beads]
7. Resuspend pellet in 30 µL of PCR-grade water. You can also use Illumina RSB.
?. [Purify the Second PCR using SPRI beads]
8. Place plate on magnetic stand, allow solution to clear.
?. [Purify the Second PCR using SPRI beads]
9. Remove 27 µL of clear supernatant and transfer to a new plate. Try to avoid bringing any SPRI beads with the sample.
?. 9. Measure concentration of first PCR using Nanodrop (A260) or Qubit Spectrophotometer.For qubit, use 2 µL Purified PCR per sample, use the 'DNA HS kit', and follow manufacturer's instructions.
?. [QC the second PCR.]
2. Confirm proper amplification by running a 2% agarose gel with 5 µL of purified product. Alternatively, you can run the gel on the PCR, before purification.The second PCR product should be extremely pure, as only the targeted fragment should re-amplify.
?. [Prepare NGS amplicons for sequencing using the Illumina Truseq Nano HT kit]
You now have purified, blunt, PCR product of the appropriate length, ready for adaptor ligation using off-the-shelf NGS kits. To use these kits, start with the "Adenylation step" to A-tail the blunt DNA, and then follow manufacturer's instructions.We will outline the next steps using the Illumina Truseq Nano HT kit (our preference) in the following steps.1. Begin at the "Adenylate 5' ends" step of the protocol. Mix 100 ng of purified PCR product with Illumina RSB (supplied) to a final volume of 17.5 µL.2. Add ATL mix and A-tail according to manufacturer's instruction.
?. [Prepare NGS amplicons for sequencing using the Illumina Truseq Nano HT kit]
3. Ligate with Illumina Truseq Nano HT adaptors (provided with the kit). Double purify with SPRI (included) as instructed.NOTE 1: We use home-made SPRI beads for PCR cleanup, but NOT for this step. Use the SPRI beads included with the kit ONLY.NOTE: Illumina includes >2X more adaptors than you need in a 96 sample kit. Save unused adaptors for later use!
?. [Prepare NGS amplicons for sequencing using the Illumina Truseq Nano HT kit]
4. Enrich DNA fragments with Illumina proprietary amplification mix/primers.NOTE: The primer cocktail is proprietary to Illumina, and is the limiting reagent in the 96 sample kit.
?. [QC NGS libraries]
We QC our libraries by three methods:1. Run a 2% agarose gel of your final enriched NGS libraries.Look for an absence of adaptor dimer at ~100 bp. Small amounts of residual adpator can severely affect downstream sequencing!
?. [QC NGS libraries]
2. Quantify libraries using the Qubit Spectrofluorometer.Use the "DNA HS" kit and manufacturer's instruction.
?. [QC NGS libraries]
3. Pool libraries based upon qubit and gel result.Ask your sequencing facility for pooling requirements. If all the libraries are the same size, you can pool on mass. If not, convert qubit data to molar concentration using an online calculator and pool using that value. Make sure the pool contains an equal molar amount of each library Use a manageable volume for the most concentrated sample, and put an equal mass of each library into the pool. We shoot for a pooled concentration of ~2-5 ng/µL.
?. [QC NGS libraries]
?. [Sequence NGS libraries]
Sequence libraries for Illumina MiSeq, 2x250 paired end read. Our sequencing facility performs this step.