GSM5418026: FANCF PEACSeq 10F [T6.10F]; Homo sapiens; OTHER
Follow the TIANGEN DP304-03 manufacturer's instructions About 500ng extracted gDNA was digested with NotI then cleaned up with 0.5x AMPure XP beads to remove the carryover plasmids. The gDNA fragments were retained on the AMPure XP beads, and on-beads Tn5 digestion was performed at 55C for one hour and adaptors were inserted at the ends of the fragments. The Tn5 was expressed and embedded with the adaptors in-house. At the end of the Tn5 digestion, 6uL 0.2% SDS was added to terminate the reaction. The products were purified and size-selected by 1.5x AMPure XP beads and eluted in 50uL H2O. The 21bp insertion sequence was used to enrich the editing sites (both on-target and off-target) in the NGS library preparation. In the 1st round of the nested PCR, two separate reactions were performed. Each reaction used a 20uL template in a total of 50uL volume at ~30 cycles. One used the PEAC-seq insertion sequence as the forward primer binding site and the downstream Tn5 adaptor as the reverse primer binding site. Another used the upstream Tn5 adaptor as the forward primer binding site and the PEAC-seq insertion sequence as the reverse primer binding site. 1st round product were purified by 1.5x AMPure XP beads and eluted in 23 uL H2O .  The 20 uL eluted DNA was used as the template in the 2nd round amplification in a total of 50uL volume at 17 cycles, and Illumina adaptors were added. The amplicons were purified by AMPure XP beads using 0.6x+0.25x double size selection. The library was sequenced on the Illumina Novaseq platform as paired-end 150bp.