GSM9063319: E0, P0 mys-1, CUT&Tag, replicate 1; Caenorhabditis elegans; OTHER
Approximately 100,000 embryos or L1 larvae were pelleted in ddH2O, snap-frozen in liquid nitrogen Frozen worm pellets were crushed to isolate single nuclei in MACS Buffer (10 mM Tris-HCl pH 7.4, 3 mM MgAc2, 5 mM CaCl2, 2 mM EDTA, 0.6 mM DTT, 1×Protease Inhibitor Cocktail) and were filtered through 30 µm Cell-Tric. After centrifugation (500 × g, 10 min, 4°C), pellets were permeabilized in OMNI Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% digitonin) for 15 min on ice, then washed twice with PBSI (PBS with 1× protease inhibitor). Nuclei were counted under microscope with Hoechst staining For each sample, 5 × 105 nuclei were combined with 5 × 102 Drosophila S2 cells (as spike-in control). Nuclei-spike-in mixtures were resuspended in MED Buffer #1 (20 mM HEPES, 271 mM NaCl, 2% BSA, 0.5 mM Spermidine, 0.01% Digitonin, 2 mM EDTA, 1×Protease Inhibitor Cocktail) containing 1 µL Bridged Protein A-Tn5 and 2 µg H4K16ac antibody (Abcam ab109463), and incubated overnight at 4°C. Nuclei were washed in MED Buffer #2 (MED Buffer #1 without EDTA), incubated in 10 mM MgCl2, at 550 rpm for 1 h at 37 °C for tagmentation, and then terminated by adding 10 mM EDTA. The nuclei were lysed in 500 mM NaCl, 1% SDS and protease K at 55 °C, 850 rpm for at least 2 h followed by column purification of DNA. Libraries were prepared using NEBNext® High-Fidelity 2X PCR Master Mix and Nextera N5/N7 primers for PCR ampliﬁcation according to recommended cycle number, followed by post-PCR clean-up with SPRIselect Beads and fragments distribution analysis. Biological duplicates per group were prepared for CUT&Tag and sent for sequencing on Illumina NovaSeq 6000. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of  3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300  bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.