adult head to-H2Av 2
Drosophila melanogaster
Flies were kept on standard media at 25C. 1-3 days old flies were collected and frozen in liquid nitrogen at the same time of the day. Frozen flies were stored at -80C until used for chromatin preparation. The following strains were used for ChIP experiments: 2202U2 (wild-type), elav-GAL4 (Bloomington stock no. 458), repo-GAL4 (Sepp, 2001), take-out-GAL4 (Dauwalder, 2002), UAS-EGFP-RPB3 (Yao et al., 2006), UAS-H2Av-GFP, gH2Av-GFP. Details of generating the latter two strains is available upon request. Fly strains used for sequencing experiments were backcrossed to 2202U2 at least for 4-6 generations. Heads of frozen flies were separated using 630 um and 400 um sieves. 400-600 fly heads were homogenized in homogenization buffer (HB) at 4C (HB: 350 mM sucrose, 15 mM HEPES pH 7.6, 10 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 0.1% Tween, freshly completed with 1mM DTT and Protease Inhibitor Cocktail (PIC)(Roche)). The homogenate was fixed using 1% formaldehyde for 10 minutes at room temperature. The tissue debris was filtered with 60 um nylon net (Millipore). Nuclei were collected and washed with RIPA buffer at 4C (RIPA: 150 mM NaCl, 25 mM HEPES pH 7.6, 1 mM EDTA, 1% Triton-X, 0.1% SDS, 0.1% DOC, freshly completed with PIC). For the fragmentation of chromatin a 2 step sonication was used: Branson250 (7 cycles, intensity 5, pulsing 16 sec) and Covaris sonicator (PIP175, DC20, CB20, time 4 min). After sonication debris was collected and chromatin was stored at -80C. Fragment size was checked after cross-link reversal on agarose gel. 10-15 ug chromatin was used per IP. Dynabeads protein G (Invitrogen) were equilibrated in RIPA with 1 ug/ul salmon sperm DNA and 1 ug/ul BSA. Chromatin was always pre-absorbed with beads without antibody. The cleared chromatin was incubated with antibodies overnight. The following antibodies were used for ChIP in this study: anti-GFP (goat, Ladurner lab stock), anti- RPB3 ((Adelman et al., 2006) and Ladurner lab stock), anti-RPB1 (7G5, Euromedex) anti-H2Av and anti-H2A (Leach, 2000), anti-H3 (abcam 1791). After IP, beads were washed 4-times with RIPA and once with LiCl wash buffer (250 mM LiCl, 10 mM Tris-Hcl pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5% DOC, freshly added PIC). Beads were resuspended in TE buffer and incubated overnight at 65 C. RNA was degraded using RNase A (Fermentas) 30 min at 37 C and proteins were digested with protease K (10 mg/ml) at 55C for 1.5 hours. Immunoprecipitated DNA was purified using Qiagen MiniElute columns and 3-10 ng DNA was sent for sequencing. In some cases, more technical replicates had to be pooled in order to obtain the required amounts. ChIP on embryos was carried as described previously (Sandmann et al., 2007). Briefly, 0-6 hours embryos were collected and washed with PBST 3% Triton-X, dechorionated with 3% Na Hypochlorite, fixed with formaldehyde and heptane. After quenching the fixing, the embryos were washed, frozen in liquid nitrogen and stored at -80C. Chromatin preparation from fixed, frozen embryos was continued with homogenization as described above.
ENA-FIRST-PUBLIC
2013-02-15T17:02:15Z
ENA-LAST-UPDATE
2018-03-08T16:46:26Z
External Id
SAMEA1710854
INSDC center name
GeneCore EMBL
INSDC first public
2013-02-15T17:02:15Z
INSDC last update
2018-03-08T16:46:26Z
INSDC status
public
StrainOrLine
to-GAL4, UAS-H2Av-GFP
Submitter Id
E-MTAB-1117:adult head to-H2Av 2
age
2 day
broker name
ArrayExpress
common name
fruit fly
developmental stage
adult
organism part
head (fat body)
sample name
E-MTAB-1117:adult head to-H2Av 2
scientific_name
Drosophila melanogaster