title: GSM4306492: Cremer-2019-RepliSeq001; Homo sapiens; OTHER
library_construction_protocol: cells were incubated with 20 µM Lovastatin (Mevinolin) (LKT Laboratories M1687) for 24 hours to synchronize in G1. 500 µM auxin or DMSO was added 6 hours before release from lovastatin block. To release from G1 block, lovastatin was washed away with 3 washes of PBS and warm media plus 2 mM Mevalonic acid (Sigma-Aldrich M4667) and 500 μM Auxin or DMSO. Cells were released for 10, 14, 18, and 22 hours. 2 hours before the time point 100 μM BrdU was added to label nascent replication. After fixation, equal numbers of cells from each release time point were pooled together for early/late repli-seq processing standard Illumina library construction protocol, Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 8-12 cycles and library fragments of 400-600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the NovaSeq following the manufacturer's protocols
attributes.tag: GEO Accession
attributes.value: GSM4306492