Illumina HiSeq 2500 paired end sequencing; Micro-C of Drosophila embryos to investigate chromatin conformation during dorsal-ventral patterning
Micro-C of Drosophila embryos to investigate chromatin conformation during dorsal-ventral patterning
We adapted the fixation and sorting procedure described in (Blythe and Wieschaus, 2015) with modifications for Micro-C. Following the first crosslinking with formaldehyde, the reaction was quenched with 2M Tris-HCl pH7.5 (0.75M final). Embryos were washed with PBST, and a second crosslinking step was carried out using long crosslinkers DSG and EGS (Sigma) at 3mM final in PBST, for 45mins at room temperature with passive mixing. The reaction was quenched again with Tris-HCl pH7.5 at 0.75M final for 5mins. Embryos were washed, sorted under a microscope, snap frozen with liquid nitrogen, and stored at -80C. Nucleic acid extraction was conducted as in Hsieh et al. 2020 with minor modifications. At least 300 nc14 embryos were used per library. Embryos were crushed in the eppendorf tube with liquid nitrogen cooled plastic pestles using 500uL buffer MB1 (50mM NaCl, 10mM Tris, 5mM MgCl2, 1mM CaCl2, 0.2% NP-40, 1X PIC). Chromatin was digested with a pre-determined amount of Micrococcal Nuclease (Worthington Biochem) to yield 90% monomer vs 10% dimer given the appropriate number of embryos. When embryos were limited, as in gd7 libraries, size selection of di-nucleosomal DNA was not carried out via gel-extraction. Library construction was conducted as in Hsieh et al. 2020 with minor modifications. Total DNA was carried over into the final library construction phase, and finally size selected for the appropriate di-nucleosomal band (350-500bp) on a 3.5% NuSieve agarose gel after PCR.