GSM8321903: CAF1degronMinus_60m_B1_T2; Mus musculus; OTHER
To the methylation reaction, 2.65 uL 10% SDS and 2.65 uL of 20 mg/mL Proteinase K (Thermo Scientific AM2548) were added and incubated at 65oC for at least 2 hours and up to overnight. To extract the DNA, an equal volume of Phenol-Chloroform was added and mixed vigorously by shaking. The samples were then spun at max speed for 2 min and the aqueous portion was removed. To the aqueous extraction, 0.1x volumes of 3M NaOAc, 1 uL GlycoBlue, and 3x volumes of cold 100% EtOH were added, mixed by inversion and incubated overnight at -20oC or for 2 hours at -80oC. Samples were then spun at max speed for 30 minutes at 4oC, washed with 500 uL 70% EtOH, spun again at max speed for 2 minutes at 4oC. The resulting pellet was air dried and resuspended in 40 uL EB. Purified DNA from mESCs and K562s was sheared using a Covaris g-tube (520079) in a 5424 rotor at 7,000 RPM for 6 passes for a target size between 6,000 and 8,000 bp. Sheared DNA was used as input for the PacBio HiFi SMRTbell Library Preparation protocol using the SMRTbell Express Template Prep Kit 2.0. Briefly, samples underwent removal of single stranded overhangs, DNA damage repair, end-repair, A-tailing, barcoded SMRTbell adapter ligation, and exonuclease cleanup according to manufacturer's protocol followed by a 1X AMPure PB bead cleanup. Final sample concentration was measured via Qubit High Sensitivity DNA Assay and library size was measured on an Agilent Bioanalyzer DNA chip. Libraries were sequenced on PacBio Sequel II 8M SMRTcells.