title: GSM6703439: Fub1_rep2; Drosophila melanogaster; OTHER
library_construction_protocol: Embryos were collected in nylon mesh sieves, dechorionated for 2 mins in 3% sodium hypochlorite, rinsed with deionized water, and transferred to glass vials containing 5 mL PBST (0.1% Triton-X in PBS), 7.5 mL N-heptane, and 1.5mL fresh 16% formaldehyde. Crosslinking was carried out at room temperature for exactly 15 mins on an orbital shaker at 250rpm, followed by addition of 3.7 mL 2M Tris-HCl pH7.5 and shaking for 5 mins to quench the reaction. Embryos were washed twice with 15 mL PBST and subjected to secondary crosslinking. Secondary crosslinking was done in 10mL of freshly prepared 3mM final DSG and ESG in PBST for 45 mins at room temperature with passive mixing. Reaction was quenched by addition of 3.7mL of 2M Tris-HCl pH7.5 for 5 mins, washed twice with PBST. Embryos were snap-frozen and stored at -80℃ until library construction.  Micro-C libraries were prepared as previously described (Batut et al. 2022) with the following modification: we used 50uL of 12-16hr embryos, non-sorted for each biological replicate. 60U of MNase was used for each reaction to digest chromatin to 80% mononucleosome vs 20% dinucleosome ratio. Libraries were barcoded, pooled and subjected to paired-end sequencing on an Illumina Novaseq S1 100 nt Flowcell (read length 50 bases per mate, 6-base index read). 