GSM3768812
GSM3768812: Δflo8_0.05_rep1; Komagataella phaffii; FAIRE-seq
The formaldehyde-fixed cells were thawed and resuspended in 1 mL lysis buffer and transferred to 2 mL screwcap tubes containing 500 µL of acid-washed 500 µm glass beads. Cells were lyzed in a FastPrep®-24 equipment (MP Biomedicals) for 5X 40 sec cycles, with 2 min rest in between. The lysate was transferred to a 15 mL conical tube. The glass beads were washed with an additional 600 µL lysis buffer which was then added to the same conical tube. The lysate was sonicated in a Bioruptor® Plus (Diagenode) for 10 cycles (30 sec on/ 30 sec off) at high power setting. The sonicated lysate was clarified by centrifugation at 10,000X g for 5 mins and around 150 µL of this clarified lysate was removed for preparation of input control DNA. The remaining lysate was aliquoted into fresh 1.5 mL tubes. 1 volume of Phenol/Chloroform/Isoamyl alcohol was added to them, vortexed vigorously and centrifuged at full speed for 10 mins and the top layer was transferred to a fresh tube. This was repeated one more time after which 200 µL of Chloroform/isoamyl alcohol was added to each tube to remove any remnant phenol. The tubes were vortexed, centrifuged and the aqueous layer transferred to a fresh tube. 1/10 volume 3M sodium acetate (pH 5.2), 2 volumes 95% ethanol and 1 µL 20 mg/mL glycogen was added to each tube and the tubes were incubated at -80°C overnight. The tubes were centrifuged at full speed for 20 mins to precipitate the DNA, after which the pellets were washed with 75% ice-cold ethanol and centrifuged again for another 10 mins. Finally, the ethanol was removed and the pellets dried and resuspended in 50 µL 10 mM Tris-HCl (pH 7.4). These FAIRE samples were treated with DNase-free RNase A (30 mins, 37°C), Proteinase K (1 h, 55°C) and finally incubated overnight at 65°C for decrosslinking. For preparation of input control DNA, the 150 µL clarified lysate removed after sonication was first treated with DNase-free RNase A, Proteinase K and decrosslinked overnight at 65°C before proceeding with Phenol/Chloroform/Isoamyl alcohol extraction as described for the FAIRE samples. Both the FAIRE DNA and input control DNA were further purified using MinElute PCR purification kit (Qiagen) and quantified. NEB DNA library preparation kit
GEO
GSM3768812
GEO Accession
GSM3768812