title: GSM9186166: SMC3_ICM_rep2; Mus musculus; OTHER
library_construction_protocol: For Adnp knockout, three single guide RNAs (sgRNAs) targeting the last exon were designed. To assemble Cas9–sgRNA ribonucleoproteins, 200 ng μl−1 sgRNAs was mixed with 0.3 μl of Alt-R S.p. HiFi Cas9 protein (Integrated DNA Technologies, 1081060) and incubated at room temperature for 15 min in 15 μl assembly buffer (1 mM Tris-HCl pH 7.5, 0.1 mM EDTA). The injection mix was centrifuged at 12,000g for 10 min, and the supernatant was used for injection into PN3 zygotes. Embryos were then cultured in G1 medium (Vitrolife). Blastocyst-stage embryos were collected for RNA-seq, CUT&RUN and Hi-C. For DRB treatment, 8C embryos (68–70 h after hCG injection) were collected and cultured in CZB medium supplemented with DRB (final concentration 120 μM) for 2 or 4 h.For the cohesin-KD experiment, PN3 zygotes were injected with 20 μM siRNAs (targeting Rad21 (Rad21 KD) or Nipbl, Smc3 and Rad21 (triple KD; Supplementary Table 2). The PN3 zygotes in the control group were injected with an equal volume of 20 μM scramble siRNAs. Then the embryos were cultured in G1 medium (Vitrolife) for 45 h. Heatshock was induced by placing the embryos or mouse ES cells at 43 °C for 30 min, followed by a recovery at 37 °C for 30 min. Control embryos or mouse ES cells were maintained at 37 °C throughout culture and were collected at the same time with heat-shock samples. Blastomere numbers and cell numbers were counted to allow downstream spike-in normalization. Embryos without the zona pellucida were transferred into 1.5-ml conventional tubes. Cells were attached to 10 μl pre-washed concanavalin A-coated magnetic beads to allow simple handling. Samples were then incubated with 50–100 μl antibody buffer (CTCF antibody (Active Motif, 61311), SMC3 antibody (Abcam, 9263) and NIPBL antibody (Bethyl Laboratories, A301-779A) diluted at ratio of 1:100) at 4 °C on a Thermomixer for overnight at 400 r.p.m. Samples were washed twice with washing buffer before being incubated with pA-MNase (700 ng ml−1 in washing buffer) at 4 °C on the Thermomixer for 3 h at 400 r.p.m. and then washed twice and resuspended in 100 μl washing buffer. Targeted digestion was performed on ice for 30 min after adding 2 μl 100 mM CaCl2 and stopped by adding 100 μl stop buffer (containing 2 μl diluted yeast spike-in DNA). Cleaved fragments were then released to diffuse out of the nuclei through incubation at 37 °C for 20 min. After removing the intact nuclei by centrifugation, the super- natant containing released chromatin fragments was used to extract DNA directly for library construction. Purified DNA was subjected to Tru-seq library construction using a NEBNext Ultra II DNA Library Prep Kit from Illumina (NEB E7645S).
identifiers.namespace: GEO
identifiers.id: GSM9186166_r1