GSM5070521: CUTRUN-AMYB-pachyteneCell-rep1; Mus musculus; OTHER
In brief, FACS-sorted germ cells were lysed using nuclear extraction buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.1% Triton X-100, 20% Glycerol) and centrifuged at 600g for 3 min at 4°C to isolate nuclei. Next, isolated nuclei were immobilized on BioMag Plus Concanavilin A coated magnetic beads (Bangs Laboratories, BP531) at room temperature for 20 min. After immobilizing nuclei to magnetics beads, nuclei were blocked using blocking buffer (20mM HEPES-KOH pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, 2mM EDTA) at room temperature for 5 min. Thereafter, 2.5 µg anti-AMYB antibody (Sigma, HPA008791, lot # C105987) was added to the beads and incubated at 4°C for 4 h using end-over-end tumbler. Of note, as negative control, we performed CUT and RUN reaction without antibody. After antibody incubation, beads were washed twice in wash buffer (20mM HEPES-KOH pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA) and incubated with 1.5 µl protein A-MNase (pA-MN; 140 µg/ml) at 4°C for 2 h using end-over-end tumbler. To remove unbound pA-MN, beads were washed twice in wash buffer. To cleave DNA beneath TCFL5 or A-MYB, 3 µl CaCl2 was directly added to the beads and incubated at 0°C for 7 min. The cleavage reaction was then stopped by chelation in 2× STOP buffer (200mM NaCl, 20mM EDTA, 4mM EGTA, 50 µg/ml RNase A, 40 µg/ml glycogen). To release the CUT and RUN fragments from insoluble nuclear chromatin, beads were incubated in 2× STOP buffer including RNase A at 37°C for 20 min and centrifuged at 16,000 g at 4°C. The supernatant containing CUT and RUN fragments were transferred to fresh 1.7 ml tube. Finally, we extracted DNA by phenol:chloroform:isoamyl alcohol (25:24:1) (pH 8) Library construction for anti-AMYB DNA was performed after DNA extraction as previously described (Zhang, Z., Theurkauf, W.E., Weng, Z., and Zamore, P.D. (2012). Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence 3, 9.).