title: GSM4262935: HomoSap.RepTiming.0hr.Replicate2; Homo sapiens; OTHER
library_construction_protocol: DNA extraction: As RerepSeq selectively fragments rereplicated DNA and enriches those sequences by size selection, it is imperative that the genomic input DNA is of high molecular weight. For yeast samples: cells were pelleted by centrifugation at 3000 rpm for 3 minutes and resuspended in SCE (1M sorbitol, 100 mM sodium citrate, 10 mM EDTA pH 8.0); fresh 0.125% (v/v) β-mercaptoethanol and 10 U/mL zymolyase was added and incubated for 30-60 minutes at 37ºC to digest cell walls; human cells or yeast spheroplasts were pelleted by centrifugation at 3000 rpm for 3 minutes and resuspended in 500uL RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF) with RNase A, 0.2 mg/mL and incubated at 37ºC for 1 hour. 25uL 20% SDS and 10µl of 20 mg/ml proteinase K was then added and allowed in incubate at 55ºC for 2 hours. DNA was extracted twice using Phenol:Chloroform:Isoamyl alcohol (25:24:1, v/v) and precipitated using isopropanol.  DNA was resuspended and stored in nuclease free water. RerepSeq Digestion: In an 8 strip 200 µl PCR tube 1 – 5 µg (depending on the experiment) high molecular weight genomic DNA was mixed with 2.5uL 10X Hoechst 33258 (0.1mg/ml) and 2.5uL 10X NEB Buffer 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 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 a Stratalinker.  Following UVA treatment,  samples were digested with 0.5uL UDG (5 units of Uracil-DNA Glycosylase), 0.5uL APE1 (10 units of human apurinic/apyrimidinic endonuclease 1) for 2 hours at 37C. Digested DNA was repaired with NEB's FFPE DNA Repair Mix for 30 minutes then separated on 0.8% agarose gel for 15 minutes.  Fragmented DNA ranging from 0.1Kb to 3Kb was gel extracted with Wizard® SV Gel and PCR Clean-Up System (Promega) and the purified DNA was resuspended in 50uL of water for subsequent qPCR and sequencing analysis. Illumina Nextera DNA Flex Library Prep Kit
attributes.tag: GEO Accession
attributes.value: GSM4262935