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

Mz F: Cel Ctct. Contot at Ha Gffi fta@tlDn@
were t ozen down at -70.C or lsed direclly icr RNA sxkadion. Each
cell lysale (sri! rri) was supp e.rrenled with 50 ,rl 2N,4 sodium scelate, pH
4.0, extracted with 5o0 /ltoi phenot (equitibfated with 10 mtv Trs and 1
mlt €OTA) a.d 100 /lo1 isoamyt6tcohot. Tubos were vortexed tor 10
seconds aod incubatBd on ic€ tor 15 minutes. Tubes we.6 spln down
in a micro.cenrituge ar 14,000 rpm tor l0 mindes at 4.c to separate
the organc and aqueous phases. Th€ aqueous phase was ransrerred
lo anoth€rtube and t ht ot isopropanotwas added ro precipirale aNA.
The samplas were tetr in -20.c to. 1 to 18 hr and th€ FNA was
corrected as a pellet by splnning rhe tLrbss in fte microcentrifuge tor 10
mrnures at 4.c. The FNA pe ers wers washed with 75% ethanot and
RNA was dissolved in 360 pt of nucieas€ tree H2O wtlh potyainyl
suphale (smg/rnD. Then, 40 lrt ot 10 x DNase I b!ffer (T s ph 8.0, 40
mrvl; NaCl 10 mMi Mgctz 6, mM) snd 20 d ot nuctease lrce ONase I
(P'omega) 1000 units/mtwer€ added. Tho rubes wer6 incubated in a
waler bath ar 37.C tor 30 m'nures Tubes we.e removed aftor the
incubarion and eilfacred wirh 4@ ut at 2s:2at g/v/v)
phenol/chlorolom/ soamyl atcohot mtxture. The tubes were spun
down and rrre supernatant was trgnsferied lo anonef tube. The
sampes w€re exrfacted onc€ with chloroior. Fotowjng ehanol
pr€optauon, the RNA pottet was washed ons wilh 750,6 ethanot and rhe
linal pellet was dissolved in 50 /t of ^uclease iree warer wilh 5 mg/ml
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