Prime it 2 random primer labelling kit

Total DNA from muscle (300 ng) as above was restricted using 10 U of BamHI‐HF or PvuII‐HF (NEB) as indicated for 1 h at 37°C. For experiments including T7 endonuclease I, 250 ng of muscle DNA was restricted using 10 U of BamHI for 30 min at 37°C, then 1 U of T7 endonuclease I (NEB) was added and the reactions were incubated for a further 30 min at 37°C. Reactions were separated on 0.6% agarose gels for 4 h at 110 V. Gels were incubated in depurination buffer (0.25 M HCl) for 20 min, followed by denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 2 × 10 min and then neutralisation buffer (0.5 M tris–HCl (pH 7.4), 1.5 M NaCl) for a further 2 × 10 min. Gels were blotted onto nylon membrane (Hybond‐N+, Amersham) overnight and then crosslinked using UV at 1200 mJ/cm². A probe (corresponding to mtDNA loci nt. 16262‐128) was synthesised by radiolabelling a PCR product using a Prime‐It II random primer labelling kit (Agilent); see Appendix Table S1 for primer sequences. Membranes were hybridised overnight at 60°C in hybridisation buffer (0.25 M phosphate buffer, 7% (w/v) SDS), then washed for 3 × 20 min with 1 × SSC containing 0.1% (w/v) SDS and imaged using a Typhoon FLA 9500 or exposed to X‐ray film.

DNA (≥10 µg) was digested overnight with appropriate restriction enzymes and run on a 0.8% agarose gel to separate fragments. Gels were subject to depurination and neutralisation before transferring DNA to Hybond-XL membrane (RPN203S, GE Healthcare Amersham) overnight by capillary action. The membrane was then washed and dried at 80 °C for 2 h. αP32–dCTP probes were labelled using Prime-It® II Random Primer Labelling Kit (300385, Agilent) and purified with Illustra ProbeQuant™ G-50 Micro columns (28-9034-08, GE Healthcare Amersham). Following pre-hybe for 1–2 h in Rapid-hyb™ Buffer (RPN1636, GE Healthcare Amersham), the membrane was hybridised with the probe for at least 5 h, washed, exposed to Fuji RX X-ray film and the film scanned on an Epson Perfection V500 Photo flatbed scanner.

Exon7 of Xist was probed using the Prime-It II Random Primer Labelling Kit (Agilent Technologies) and Cy3-dCTP (GE). The RNA-FISH experiments were performed as previously described [35 (link)]. We conducted the Fisher’s exact test and Chi-square test to examine differences in cells with Xist clouds between developmental stages.