PCR amplification and next generation sequencing of sgRNAs

1. Amplify DNA fragments containing sgRNA sequences from isolated genomic DNA by two rounds of PCR using NEBNext Ultra II Q5 master mix (NEB, Ipswich, USA). For the first round of PCR, perform forty-eight 50-μL PCR reactions (each containing 2 μg of genomic DNA template) with the forward primer NGS-Lib-KO-Fwd-1 (5’- CCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNGCTTTATATATCTTGTGGAAAGGACGAAACACC -3’) and the reverse primer NGS-Lib-KO-Rev-0 (5’-CAGACGTGTGCTCTTCCGATCTCCGACTCGGTGCCACTTTTTCAA-3’) with a thermal cycler program consisting of initial denaturation at 98°C for 5 minutes, followed by 16 cycles of (98°C denaturation for 10 seconds, 69°C annealing for 30 seconds, and 65°C extension for 45 seconds), and a final extension at 65°C for 5 minutes. 
2. Pool and purify the products of the first-round PCR by a DNA clean and concentrator kit (Zymo, Irvine, USA) and dilute to 2 ng/µL.
3. For the second round of PCR, perform six 50-μL PCR reactions (each containing 2 ng of the purified first-round PCR product) for each sample with the forward primer NGS-Lib-KO-Fwd-2 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and one of four indexed reverse primers (NGS-Lib-KO-Rev-1, 2, 3, 4) (5’-CAAGCAGAAGACGGCATACGAGAT-8-nucleotide index-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’). Use the same cycling conditions for second-round PCR as the first-round PCR for 18 cycles. 
4. Run the products of the second-round PCR reactions on a 2% agarose gel. 
5. Excise the expected ~300 bp amplicons and extract DNA from the gel for next generation sequencing (Illumina Hiseq PE150).

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