Pcr 05 c

The fixed single blastocyst was transferred to a PBS drop (1 μL) in a 0.5 mL PCR tube (#PCR-05-C.; AxyGen Scientific, Inc., Union City, CA, USA) with the help of a mouth piece-controlled micropipette, as previously described [33 (link),34 (link)]. Genomic DNA was extracted by adding 100 μL of lysis buffer mentioned above and lysed at 37 °C for over two days. The solution was extracted with phenol/chloroform [43 (link)] and the supernatant was precipitated with isopropanol. The precipitated DNA was dissolved in 20 μL of sterile water and stored at 4 °C. To increase the amount of whole genomic DNA, we employed WGA using illustra GenomiPhi V2 DNA Amplification Kit (#25-6600-31; GE Health Care Japan, Tokyo, Japan), as previously described [34 (link),43 (link)]. Briefly, 2 μL of genomic DNA was mixed with 8 μL reaction buffer containing enzyme in a 20 μL volume and allowed to react overnight at 30 °C. The resulting WGA products (2 μL) were subjected to the first PCR as described above.

Sorted cells were centrifuged in a swing-out rotor at 4,000 g for 15 min at 4°C, transferred to a thin-walled 0.5 ml microfuge tube (Axygen PCR-05-C), re-centrifuged and then resuspended in 130 μl Lysis Buffer (17 mM Tris.HCl pH 8, 3.4 mM EDTA.Na₂, 0.34% SDS) containing protease inhibitors (Sigma-Aldrich P8340). The lysate was sonicated for 5 cycles at high setting using a Diagenode Bioruptor (1 cycle is 30 sec ON and 30 sec OFF). After sonication, the sample was centrifuged at 16,000 g for 15 min at 4°C, the chromatin-containing supernatant transferred to a fresh microfuge tube and 70 μl RIPA Buffer (36.7 mM Tris.HCl pH 8, 2.5 mM EDTA.Na₂, 0.01% SDS, 2.46% Triton X-100, 374 mM NaCl) containing protease inhibitors added to the chromatin sample. The ChIP reaction, washes and DNA purification were performed as in Dahl and Collas [56 (link),57 (link)]. In brief, magnetic beads were coated with 2.4 μg of rabbit anti-H3K27me3 antibody (Millipore 07–449) and incubated overnight in a volume of 100 μl with chromatin from ~100,000 YFP⁺ sorted cells. Beads were washed, chromatin eluted, RNA and proteins digested, the DNA purified by phenol/chloroform extraction followed by ethanol precipitation using linear acrylamide as carrier and resuspended in 10 μl PCR grade water. Approximately 5 μl of chromatin was retained as input and purified alongside the ChIP sample.