Epigenetic regulation of reactivation of the silenced X-chromosome during mouse germline development
Epigenetic regulation of reactivation of the silenced X-chromosome during mouse germline development
Experimental F1 embryos were generated by crossing Xist+/∆ (B6) dams carrying Oct4-EGFP transgene, and CAST sires, producing F1 female (XX(Xist∆)) and males (XY) that were analyzed.  Following timed natural matings, F1 embryos were harvested daily at E13.5 and E16.5. Gonads were dissected and dissociated into single-cell suspension. EGFP fluorescence was used to isolate the fluorescently marked germ cells with FACS. Viable individual germ cells and somatic cells were sorted by FACS, and bulk sorting was performed to collect purified germ cell and / or somatic cell fractions separately into 300μL of collectionbuffer (5% BSA-HBSS) and used fresh. please see nucleic acid library construction protocl For each CUT&RUN reaction, typically ~5000 cells were used, pooled from different embryos of identical genotype in the same litter. Here we applied a CUT&RUN method modified from previous protocol (Derek Janssens, Steven Henikoff 2019. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io https://dx.doi.org/10.17504/protocols.io.zcpf2vn). Sorted cells were washed with Wash Buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine and a Roche complete tablet per 50 ml), and then bound to activated Concanavalin A (ConA) -coated magnetic beads. After 10mins of binding, cells were permeabilized and incubated in 50μL Antibody Buffer (Wash buffercontaining 0.01% Digitonin and 0.5μL of specific primary antibody) at 4°C overnight on a nutator. For the negative control, anti-Rabbit IgG was added instead of primary antibody. Next day, after 2 washes with Digitonin buffer (Wash Buffer containing 0.01% Digitonin), beads were resuspended in pAG / MNase buffer (50μL Digitonin buffer with 2.5μL of pAG / MNase reagent (EpiCypher) and incubated at room temperature (10 mins) on a nutator for pAG-Mase binding. After washing away pAG / MNase with Digitonin buffer, the chromatin digestion was performed by adding 1μl of 100mM CaCl2 to each reaction and incubating at 4°C for 2 hours on a nutator. Digestion was then stopped with the addition of 33μL Stop Buffer (340 mM NaCl, 20mM EDTA, 4mM EGTA, 50μg / mL RNase A, and 50μg / mL glycogen) and the cleaved chromatin was released to supernatant by incubating at 37°C for 10 mins. DNA was then purified using a DNA clean-up column (Monarch® PCR & DNA) following the manufacturer's instructions, and used for library preparation (NEBNext® UltraTM II DNA Library Prep Kit for Illumina®). Specifically, the concentration of adaptor (reduced to 3μM) and the cycle number of PCR amplification (15 cycles) were optimised in our experiment considering of the low input of DNA. Finally, the clean-up of PCR product was carried out by using AMPure X beads (Beckman Coulter) (in a ratio of 0.9:1). We determined the specificity for the antibodies used, using the SNAP-CUTANA K-MetStat Panel (EpiCypher). 2μL of 1:20 diluted solution was applied to ConA-immobilized cells prior to the addition of antibody. As per the manufacturer's instructions, reads were matched to the unique DNA barcodes in the panel and normalized to either on-target or total counts.