GSM5051711: noCopperWGS_rep2; Saccharomyces cerevisiae; OTHER
Rerep-seq was performed as described in . Briefly, In an 8 strip PCR tube 1 – 5 ug high molecular weight genomic DNA was mixed with 2.5uL 10X Hoechst 33258 (0.1mg/ml) and 2.5uL 10X NEB 4 (50 mM Potassium Acetate, 20 mM Tris-acetate , 10 mM Magnesium Acetate, 1 mM DTT pH 7.9@25°C) to a final volume of 24uL; open tubes placed upright in PCR tube rack, covered with a glass plate (3" x 3" glass plate from VWR Vertical Gel Electrophoresis Systems), exposed to 7.5 minutes of glass plate filtered (UVA only) from Stratalinker followed by addition of 0.5uL UDG (5 units of Uracil-DNA Glycosylase), 0.5uL APE1 (10 units of human apurinic/apyrimidinic endonuclease 1) for 2 hours at 37°C. The digested DNA was repaired with NEB's FFPE DNA Repair Mix for 30 minutes; mixed with 10X loading dye then run on 0.8% agarose gel for 15 minutes. The fragmented DNA ranging from 0.1Kb to 3Kb was gel extracted with Wizard® SV Gel and PCR Clean-Up System with the purified DNA eluted in 50uL nuclease free water. Sequencing libraries were constructed using the Illumina Nextera DNA Flex Library Prep Kit (catalog number 20018704) following the manufacturers' protocol and using 14 cycles of amplification for final library construction. Libraries were constructed using dual index barcodes from the Nextera DNA CD Indexes (catalog number 20018707).