GSM8840825: Nanos-Upd E Fraction 1 Replicate 2-PE; Drosophila melanogaster; OTHER
Whole tumor testes of the appropriate genotype were dissected in Schneider's media to obtain 100-200 pairs. The dissected testes were incubated for 15 minutes in media containing 100mM BrdU. After removing the media, nuclei were isolated by resuspending testes in 1mL homogenization buffer. Testis tissues were dounce homogenized with 20 strokes with loose pestle (A), followed by 40 strokes with tight pestle (B), before pipetting gently ~20 times with a wide bore tip. Nuclei were filtered through at 40uM cell strainer, then 10uM cell strainer into a fresh tube. The homozenization buffer was removed after pelleting nuclei. The pelleted nuclei were resuspended in resuspension buffer containing propidium iodide and incubated on ice for 30 minutes before proceeded to the cell sorter.  Nuclei were sorted on a MoFlo XDP cell sorter by gating for GSP+ nuclei and dividing S-phase nuclei into 4 equal fractions corresponding to early, early-mid, late-mid, or late replicating nuclei. Following the sort, DNA was purified from the nuclei. The purified DNA was next sonicated into ~200bp fragments, before initiating library preparion. The NEB DNA Ultra II library prep kit for Illumina was incorporated into the Repli-seq protocol.  The first two steps of the NEBNext Ultra II DNA Library Prep Kit (End Prep and Adapter Ligation) were performed according to the manufacturer's instructions. The adapter-ligated DNA fragments were cleaned using the Zymo Research DNA Clean & Concentrator-5 kit. Next,  BrdU-containing DNA was immunoprecipitated, which enriches for DNA that was actively replicating at a specific stage in S-phase. The immunoprecipitated DNA was purified using a DNA Clean & Concentrator-5 kit from Zymo Research. Efficiency of the BrdU-immunoprecipitation was calculated using qPCR of the sample DNA compared against a serial dilution of known DNA concentrations. This method determined the number of PCR cycles required for library amplification. PCR amplification of the DNA fragments was performed following the NEBNext Ultra II DNA Library Prep Kit for Illumina.