7 protocols
normalization data transformation protocol
Scanning was carried out according to manufacturer's specifications.
normalization data transformation protocol
Reads were aligned to the Drosophila melanogaster genome (BDGP5.13) using Bowtie version 0.12.7 (Langmead et al., 2009) with the parameters -n 3 -m 40 -k 1 --best --nomaqround. Aligned reads were filtered for duplicates, uncalled bases (maximum 3 Ns were allowed) and low complexity (reads with stretches of > 20 identical bases were removed). Only chromosomes 4, X, 3L, 3R, 2L and 2R were considered in the analyses. Regions of high ChIP enrichment were detected with CCAT 3.0 (Xu et al., 2010) on individual replicates using Input or ChIP against GFP and/or H3 as controls with the parameters “isStrandSensitiveMode 0, outputNum 100000, randomSeed 123456, minScore 4.0, bootstrapPass 50 “ and “fragmentSize 200, slidingWinSize 100, movingStep 20 , minCount 8“ for RNA Pol II, respectively fragmentSize 250, slidingWinSize 500, movingStep 50 minCount 20” for H2A.v. Regions occurring in more then one replicate at a reported FDR rate <=0.05 were merged with regions occurring in single replicates at an FDR <=0.01. All replicate information was used to restrain the region and gain maximum resolution. Overlapping peak summit positions, read counts and scores were averaged (see Supplementary File PeakCalling-Supplemental). Peaks were called using the same procedure in the control files and the union of the regions used to eliminate low-scoring, putative false positive peaks. Regions with a mean score < 4.5 that overlapped positive regions in the control peak calls were removed. Regions from different tissues were merged together to obtain a set of regions of interest (ROIs) for (A) the elav-repo comparison - “Head-ElavRepo”; (B) the elav-repo-to comparison - “Head-All” and (C) the head-embryo comparison - “Head-Embryo” (Supplementary File PeakCalling-Supplemental).
nucleic acid sequencing protocol
Library preparation and sequencing was performed by the EMBL Genomics Core facility using standard Illumina protocols. 36bp single run was used on the Illumina GenomeAnalyzerIIX. All ChIP-Seq was done in two biological replicates. As control, one Input lane was sequenced.
nucleic acid library construction protocol
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.
growth protocol
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.
normalization data transformation protocol
Count information in individual ROIs (A, B or C) for either RNA Pol II or H2A.v were compared using DESeq ver. (Anders et al., 2010), determining significant differences in ChIP enrichment between pairs of tissues. As a control, we used the DESeq variance estimations from pairwise tissue-comparisons to call significant differences between the replicates of the same tissue. If at the significance level of 0.01 (after multiple testing correction) over < 5% of ROIs were found to be different among replicates of the same tissue, we lowered the cutoff. We used the steps 0.005, 0.0001, 0.00005, up to 1*10-6. This approach resulted in the cutoff of 0.01 for all comparisons, except for RNA Pol II Repo-To and Elav-To, where 0.001 was used. Additionally, we required a fold-difference of at least 1.8 between estimated mean counts.