Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

A2iz F: c.u cwL contat al Ha GeN f@diDti@
constructs. RNA samples w€.€ supplemenl€d with 1/10th volum€ of 5 M
NH.OAC and 2.5 vololerhanoland mixed lhoroughlyby vodexlng. Tlie tubes
were placed in the 20'C lreozer lor 15 minules. Along with A'e expedmenial
samples, tso control lubes were p/epaed which conlain s rr9 yeast RNA
(neqallve cofrrol RNA) FNA was prgcipitated by centritlgation at maxi.num
speed 14,000 rpm in $e microlugo lor 15 mindes at 4'c. Th€ supernabnt
was caretully aspkated and dried on sofr tissue (Kimwipeg. Tubes werc
cenrrilug€d brielly to co lect residual ethanoL at rhe bonom oflhe lube, and this
r€maining fluidwas removed using a pipene man.
P€llels were dissotued in 20 ,rl oi solu{on A (hybddiz6tion bufie4.
Samples wero thrcrcughly mixed by vodelng ior 5 to 10 se€onds and then
centrltuged to colecr the liquid at lhs bottom ol rhe lubes. Tubes were
translered to 90 - 95'C wate. barh for 3 b 4 minures ro denature and
solubilize RNA in rhe bufier. Samples were lhen re-vo.texed and brieUy re,
centriiuged, owed by ncubation at 42 to 45
'ol
. C n a waterbath lor at sasr 18
hours. Ar lhe end oi lhe incubation 2O0 Al of solutio. F+AX (ANass A +
RNa* f mixlur€ in giycerol and FNA digestion builer) was added to each
reacton at a ralro ol 1:100 (v/v). ore controLtube which conra ns yeasl FNA
(r€presenting u.digesred probe) was dissolved in 200 |jl of FNA digesrion
bufier withoul lhe RNAse enzyme. The second conrrol lube containing yeasr
FNA was also lncluded wlh oach set ol samp]es (ths sample sholLd yield a
compl€rely digesred probe), The iubes wer€ vonex6d ce.trlluged and
incubated ar 37"c lor 30 minlles ro digesl lhe unprotecled po.lio.s ot lhe