Standard NextSeq 500 paired-end Sequencing; Capture Hi-C on boundary insertion in Drosophila melanogaster MiMIC lines
Capture Hi-C on boundary insertion in Drosophila melanogaster MiMIC lines
Wildtype Drosophila melanogaster flies were grown in population cages at 25°C. MiMIC fly lines with boundary insertions were grown in large-sized cages at 25°C. 0-16 hour embryo collections were aged at 25°C up to the desired time point (4-20 hours). Two independent collections were performed for each timepoint. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30 ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. ; 1g of frozen embryos were dounce-homogenized, 20 times with a loose pestle and 10 times with a tight pestle, in 10 ml of chilled HB buffer (15 mM Tris-HCl pH 7.4, 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA and 0.2 mM EGTA) on ice. The lysate was filtered through 2 layers of Miracloth and spun at 3,200g for 10 min to pellet the nuclei. The nuclei were then washed in 10 ml of chilled HB buffer, spun at 3,200g for 10 min and resuspended in 3 ml of PBTB buffer (5% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100 in PBS). The nuclei were dissociated by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe, and filtered through a 20-µm Sefar Nitex membrane. Nuclei were counted on a Symphony A3 cell analyzer. 30 million nuclei aliquots were frozen in liquid nitrogen. ; Capture Hi-C libraries were generated using a Bridge-Adaptor in situ Hi-C protocol and 75-bp capture probes, each designed on either end of a DpnII fragment. 25 million dry-frozen nuclei were thawed in 1 mL ice-cold lysis buffer for 30 min and permeabilized at 65°C for 10 min with SDS. Chromatin was digested overnight at 37°C with 8 µL of the restriction enzyme DpnII. 5 µL 90 μM annealed biotinylated bridge oligos were ligated to the fragmented DNA. Nuclei were washed in a solution with BSA and decreasing SDS concentration. To enable adaptor::adaptor ligation, nuclei were treated with PNK followed by ligation. Proteins were digested by incubation with 20 µL of 20 mg/mL Proteinase K at 55°C, 1000 rpm shaking for 30 min, followed by the addition of 130 µL of 5 M NaCl and decrosslinking on a shaker at 68°C and at 1000 rpm overnight. 63 µL of 3 M NaOAc pH 5.2, 2 µL of 15 mg/mL GlycoBlue (Life Technologies) and 1 mL of absolute ethanol were added, and the samples incubated at -70°C for 15 min.  The DNA was precipitated by centrifugation for 1 h at 20000 g at 4°C.  RNA was digested with 0.2 μg/µL RNase A at 37°C for 15 min.  The DNA was sheared ∼200-400 bp with Diagenode Bioruptor Pico (4°C, 8 cycles, 30”/90” ON/OFF), and size-selected with AMPure XP beads 1–0.6x. Biotinylated DNA fragments were captured with 30 μL of Dynabeads Streptavidin M-280 beads. The sequencing library was prepared using the Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Swift biosciences, 21024), with slight modifications to allow library preparation from DNA bound to Streptavidin beads (from the library preparation published by Arima https://arimagenomics.com/wp-content/files/User-Guide-Library-Preparation-using-Swift-Biosciences-Accel-NGS-2S-Plus-DNA-Library-Kit.pdf).  The library was amplified with eight PCR cycles, quantified by Qubit and checked on a Bioanalyzer. Samples with compatible indexes were pooled and processed according to the Twist Target Enrichment protocol, with minor modifications. After quantification with Qubit and quality control on a Bioanalyzer (displaying a single peak between 350 and 500 bp), the libraries were sent for sequencing on an Illumina NextSeq 500 platform 150 PE using standard Illumina primers.