3.2. Amplification of sgRNA or Barcode Sequences from Genomic DNA

The protocol is based on two rounds of PCR to amplify the integrated sgRNA, shRNA barcode sequences, or clonal barcodes from the genomic DNA. Also, the second round of PCR includes primers that add the sample-specific indexes and sequences (P5 and P7) complementary to primers immobilized in Illumina flow cells. With the PCR reactions, the optimization of cycles is required to avoid over-cycling. Using an optimal cycle number is necessary to generate a sufficient amount of amplicons to sequence while maintaining the relative representation of each sgRNA or barcode in the cell population.

• As an amplification/sequencing positive control, we recommend that you use the original shRNA/sgRNA/barcode plasmid library (1 ng) used in the screen. This positive control can be used as a baseline in NGS data analysis to measure the enrichment or depletion of effector constructs in the experimental samples. If you do not have the plasmid library, we suggest either using any construct in the same vector to serve as a positive control for PCR or the packaged library to serve as an amplification/sequencing positive control as described in the following bullet.

• Although it is typically not necessary, you may use the packaged effector library as a positive control. In order to use the packaged library as a positive control, you can start with a volume containing sufficient viral particles equal to at least a few hundred-fold the complexity of the library. For example, for a 50,000-construct library, you should use a volume containing 10-25 million transduction units (TU). Also, before starting PCR, you will need to reverse transcribe the viral RNA sequence using a reverse primer that targets the sense (upper) strand of the insert site at a location where it will generate a full transcript of the target sgRNA/barcode region, including the PCR primer sites. For most Cellecta libraries containing the UbiC promoter, the RevUbiC1 primer (5’-AGGCAGCGCTCGCCGTGAGGA-3’) adjacent to the cloning site can be synthesized and used for the reverse transcription step.

First Round PCR

The goal of the first PCR is to amplify shRNA barcodes, sgRNA inserts, or barcode library sequences from genomic DNA isolated from each experimental cell population. The amount of starting DNA can vary depending on the type of screen. For samples with up to 50 µg of genomic DNA, run a single 100 µl reaction for the each sample. For samples with more than 50 µg of DNA, we recommend dividing up the run into multiple 100 µl PCR reactions, each with a maximum of 50 µg DNA.

1. For each sample, prepare a PCR mix according to the table below, where x is the volume of genomic DNA (typically 50 µl at 1 µg/µl). Many samples will have more than 50 µg of DNA. For these samples, you will need to scale up the master reaction below as needed for each sample, then divide the reaction into multiple tubes with 100 µl in each. For example, if you have 180 µg of DNA in one sample, you should prepare a 400 µl master mix, then aliquot 100 µl in four PCR tubes for amplification (180 µg / 50 µg = 3.6, so round to 4 reactions).
   

   Volume	Component
   x µl	Genomic DNA (up to 50 µg)
   3 µl	Forward 1st Round PCR Primer
   3 µl	Reverse 1st Round PCR Primer
   2 µl	dNTP Mix
   10 µl	Taq Polymerase Buffer
   80 – x µl	PCR-Grade Water
   2 µl	Taq Polymerase
   100 µl	Total volume

2. Mix gently, centrifuge briefly to collect droplets, divide samples with more than 50 µg of genomic DNA into 100 µl PCR reactions as necessary.

3. Perform PCR using the following cycling conditions.
   

   95°C, 2 minutes	1 cycle
   95°C, 30 seconds, 65°C, 30 seconds, 68°C, 2 minutes	16-18 cycles*
   68°C, 2 minutes	1 cycle

* Note: Depending on your library, the specific number of cycles may need some optimization. For barcode libraries (and shRNA libraries with barcodes), 16 cycles is typically sufficient. For sgRNA libraries and dual-sgRNA libraries, 18 cycles is recommended. If you are using less than 50 µg of genomic DNA in a 100 µl PCR reaction, increase the number of cycles (e.g., for 25 µg, use one extra cycle). However, please note that we do not recommend analyzing the First Round PCR product by gel or equivalent (Bioanalyzer, etc.). You may not see a band at all or just a light band at most. You should evaluate the PCR product only after running both First and Second Round PCR.

Second Round PCR

The Second Round of PCR is required in order to significantly reduce genomic DNA carryover into the NGS step. Additionally, the Second Round PCR primers add the P5 and P7 sequences that are complementary to the immobilized primers in the NGS Illumina Flow Cells.

• For Cellecta NGS Prep Kits and Supplementary Index Primer Sets, the NRev Index Primers (up to 24) each contain a different index sequence which will be used to deconvolute the sequencing results for each sample mixed together in the same Flow Cell or sequencing lane (i.e., multiplex sequencing). It is important, therefore, to use a different NRev Index Primer (each signified by a different letter) to amplify each sample that will be run together in the same Flow Cell or lane.

For the Second Round of PCR, start with an aliquot of 5 µl of the First-Round PCR reaction for each sample.

1. If you are starting from more than 50 µg of genomic DNA, it is important to combine all individual reactions for each sample together into a single tube.

2. Perform the Second Round of nested PCR using a 5 µl aliquot of the First Round PCR product for each sample:
   

   Volume	Component
   5 µl	First Round PCR Product
   5 µl	NFwd Primer
   5 µl	NRev (Index) Primer*
   2 µl	dNTP Mix
   10 µl	Taq Polymerase Buffer
   71 µl	PCR-Grade Water
   2 µl	Taq Polymerase
   100 µl	Total volume

* Note: Each sample to be sequenced together in a single Flow Cell or lane needs to be amplified with a different NRev Index Primer in the Cellecta NGS Prep Kit. Be sure to use a differently lettered NRev Index for each sample that will be loaded on the same Flow Cell or lane.

3. Perform PCR under the following cycling conditions.
   

   95°C, 2 minutes	1 cycle
   95°C, 30 seconds, 65°C, 30 seconds, 68°C, 2 minutes	12 -16 cycles**
   68°C, 2 minutes	1 cycle

** Note: Additional cycles may be needed for samples as described below.

4. Analyze the amplified products from the Second Round of PCR. This analysis may be done on an Agilent® 2100 Bioanalyzer® instrument with the Agilent® High Sensitivity DNA Kit (Cat.# 5067-4626) or a Fragment Analyzer (Advanced Analytical) instrument using Standard Sensitivity NGS Analysis Kit (Cat.# DNF-473-1000) using the manufacturer’s protocol, if you have access to this equipment. If not, then we recommend the following procedure using agarose gel electrophoresis to analyze the samples:
   • Run all samples including positive control on 3.5% agarose-1xTAE gel. Mix 5 µl of aliquot from the each sample with 2x loading buffer (e.g. 10% sucrose with Bromophenol Blue tracking dye).
   • Analyze the intensity of the bands after electrophoresis (i.e., Bromophenol Blue tracking dye about 80% down the gel). The typical results of agarose gel electrophoresis analysis are shown in the figure below. The expected size of amplified barcode, shRNA barcode, and sgRNA products is typically between 125 to 400 bp or dual-sgRNA product around 750bp and is provided in the NGS Cassette Design information specific for your library or NGS Prep Kit Product Certificate. If you did not receive Cellecta’s Order Information email with links to this information, please contact us at tech@cellecta.com.

5. Quantify the amount of the amplified product for each sample using software tools provided with instrument or gel imager. If all samples show similar yield of amplified products (+/- 2-fold) no addition cycling is required and you should proceed to NGS Samples Purification. If there is a greater than 2-fold variance, you should put the samples with very weak or missing bands back into the PCR instrument for additional cycles. For example, if the amount of PCR product in the sample is 4, 8, or 16-fold less than in other samples, run that sample for an additional 2, 3, or 4 PCR cycles, respectively. For samples in the +/- 5-cycle range, don’t run additional cycles.
   • If you run additional cycles on any samples, quantify the reaction products again by using one of the instruments mentioned above or gel electrophoresis to ensure that all samples have correctly amplified products.
   • Avoid overcycling! Try to use the minimum number of cycles which allows quantitation of the bands. Importantly, for dual-sgRNA libraries, overcycling induces a significant level of recombination between different dual-sgRNA molecules. Overcycling also often generates additional higher molecular weight bands. These bands do not normally interfere with NGS unless they are very strong. Also, overcycling has the potential to compress the representation of the targeted sequences if the reaction exceeds the exponential phase. Therefore, it is desirable to minimize the cycle number. If there are excessive higher molecular weight bands, you can rerun Second Round PCR for that sample using another 2 µl of DNA from the First Round PCR and a fewer number of cycles.

DNA samples can be stored for several days at +4°C at this stage or move directly onto NGS Sample Purification

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