GSM5026037: DAP_lib1_GUS_100ngDNA; Arabidopsis thaliana; OTHER
DNA was extracted with DNeasy Plant Mini kit (Qiagen) The genomic DNA was diluted with water into 55 µl aliquots containing 1000 ug of DNA each and sheared with the Covaris S2 instrument to an average fragment size of 200bp (Duty Cycle = 10%, Intensity = 5, Cycles per Burst = 200, Treatment time = 120 seconds). 50 µl of sheared DNA underwent steps 1 to 3 of library construction using the KAPA Hyper Prep kit (KR0961) (End Repair and A-tailing, Adapter Ligation, Post-ligation Cleanup). The adapter ligation was performed using 5 µl of Y adapters, which were annealed as described in the published DAP-seq protocol (PMID 28726847). The Post-ligation Cleanup was done with 1X AMPure beads (Beckman Coulter) instead of 0.8X. The libraries were eluted in 20 µl of EB buffer, pooled according to genotype, and quantified with the Qubit instrument. The rest of the DAP-seq protocol was performed as described in PMID 28726847 using 1000ng of plasmid DNA for each TNT expression reaction, and with either 15, 30, or 100 ng of genomic DNA, as indicated in the sample names.  The final PCR amplification of the pulled-down DNA was performed using the Fusion High-Fidelity PCR Master Mix with HF Buffer (F531S) in a final volume of 60 ul, with the following cycling parameters: 95°C for 2 min, 20 cycles of [98°C for 15 sec, 60°C for 30 sec, 72°C for 1 min], 72°C for 10 min. Each sample was individually run on agarose gel and the smear between 200 and 400 bp was cut out, purified and sequenced.