title: brt20_c4_RVBarcode
design: To enrich the barcodes from DNBC4 cDNA library, 1 μL of cDNA with 50 μL PrimeSTAR Max, 10 μL of 10 μM MGI-F21-AD primer, 10 μL of 10 μM mRuby-linker-F, and the volume filled up to 100 μL with nuclease-free water. First-round amplification was carried out at 98 ℃ for 5 minutes, followed by 10 cycles of 98 ℃ for 30 seconds, 58 ℃ for 30 seconds, 72 ℃ for 60 seconds, with a last extension at 72 ℃ for 5 minutes, and finally ended with 4 ℃ forever. PCR products were purified using 1.2x DNA clean beads. Primers used in the first-round amplification contain adaptors for the second-round PCR to incorporate Illumina adapters. Second-round amplification was carried out at 98 ℃ for 5 minutes, followed by 5 cycles of 98 ℃ for 30 seconds, 58 ℃ for 30 seconds, 72 ℃ for 60 seconds, with a last extension at 72 ℃ for 5 minutes, and finally ended with 4 ℃ forever. PCR products were purified using 1.0x DNA clean beads.