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Sample GSM1200071 Query DataSets for GSM1200071
Status Public on Aug 02, 2013
Title H3K27ac_(Active_Motif).L3.Solexa Input Rep1
Sample type SRA
Source name H3K27ac_(Active_Motif).L3 Input expt.2330
Organism Drosophila melanogaster
Characteristics strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type )
developmental stage: 3rd Instar Larvae
genotype: wild type
Extracted molecule genomic DNA
Extraction protocol Flies are grown in population cages. After the larvae mature into flies in the inoculated tubs, the flies are transferred to the population cages. The adult flies are raised over a 22-day cycle, with cycles overlapping so that when one adult population has become less productive, another is ready to take its place.
Growth conditions and collection protocol to harvest third instar larvae from bottles.
Drosophila tissues were homogenized, and treated with formaldehyde. Nuclei were lysed with SDS and sonication in the presence of protease inhibitors. The resulting chromatin was treated with non-ionic detergents at physiological concentrations of monovalent cations.
1. Add 30ml of PAS (50% suspension in RIPA (-PMSF)) to 500ml of crosslinked chromatin. Incubate 1h at +40C. 2. Spin suspensions for 2min at top speed +40C. Transfer supernatants to new tubes. Add 5ml of 100mM PMSF solution in isopropanol to each 500ml aliquot of precleared chromatin. 3. Add appropriate amount of antibody to each reaction. Do not forget to set up no Ab control. Incubate for 15 hours at +40C on rotating weel. 4. Add 40ml of PAS (50% suspension in RIPA (-PMSF)), incubate 3h at +40C on rotating weel. 5. Wash the beads 5 times 10min each with 1ml of RIPA, then one time with 1ml of LiCl ChIP buffer and finally twice with 1ml of TE. To pellet the beads between washes spin samples for 20sec +40C at top speed. Do all the washes at +40C. 6. Resuspend the beads in 100ml TE add 1ml (final 50mg/ml) of RNAse A (10mg/ml) incubate 30min at +370C. 7. Add 7.5ml (final 0.5%) of 10% SDS and 3.8ml (final 0.5mg/ml) of Proteinase K (20mg/ml). Incubate overnight at +370C. 8. Transfer samples at +650C, incubate 6h. 9. Add 4.5ml of 5M NaCl (140mM final). Extract samples with 150ml of phenol-chloroform by vortexing for 30 sec, centrifuge for 10 min at RT, take 120ml of aqueous phase, back-extract organic phase with 150ml of TEN 140 (10mM Tris-HCl pH8.0; 1mM EDTA; 140mM NaCl). Take 150ml of aqueous phase. Combine aqueous phases (you will get 120ml + 150ml=270ml of solution). 10. Extract samples with 300ml of chloroform by vortexing for 30 sec, centrifuge for 5 min at RT. Transfer the upper aqueous phase into the new tube. Add 30ml of 3M NaAc pH 5.0 and 2ml of glycogen (5mg/ml) to aqueous phase. Precipitate DNA with 900ml of EtOH at -700C for 1h. 11. Spin for 10min, top speed at +40C. Wash the pellet in 300ml of 70%EtOH 12. Spin for 10min, top speed at +40C. If you plan to do qPCR analysis only dissolve the pellet in 150ml of pure H2O. If you plan to do both qPCR and microarray hybridization then first dilute DNA pellets in 12ml of pure H2O transfer 4ml of DNA solution to a new eppendorf tube and add 46ml of pure H2O. Use the latter for qPCR and the former for subsequent amplification and labeling. Store DNA solutions at -200C. 13. Prepare dilutions of DNA isolated from the original crosslinked chromatin (also called ?Input DNA? or simply ?Input?) following the chart below. Use the stock with concentration of 0.5% of input DNA per ml of solution (see: ?Isolation of ChIP Input DNA? protocol).
Protocol for making libraries for ChIP-seq with Illumina platform, using the TruSeq DNA Sample Prep Kit.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
Description input DNA
Data processing Bowtie alignment protocol. Bowtie is an ultrafast, memory-efficient short read aligner geared toward quickly aligning large sets of short DNA sequences (reads) to large genomes.Reads that passed default parameters of the Illumina quality filter were mapped with Bowtie 0.12.7. The reference sequence used to built an index for mapping was the Flybase reference r5.22. The reference index was created using the bowtie-build function with default parameters. Reads were mapped using -n 2 -l 28 -e 70 -k 1 -m 10 --best in Bowtie (-n 2, -l 28, -e 70 default). Parameters-n INT number of allowed mismatch-l INT seed length-e INT maximum permitted total of quality values at all mismatched read positions-m INT suppress all alignments for a particular read or pair if more than INT reportable alignments-k INT report up to INT valid alignments--best report alignments in best-to worst orderReads were converted to SAM formant using the script provided by the DCC. SPP Peak calling protocol. The aligned reads were called using SPP R package (ver.1.10, Kharchenko et al., Nat.Biotech 2008). The binding characteristics and tag shift were estimated using SPP get.binding.characteristics() function. The reads showing abnormality were removed using SPP remove.local.tag.anomalies() function. The regions of enrichment were identified as continuous blocks of positions that exhibit significant enrichment of IP over input reads. The significance of enrichment was assessed within a 1Kb window around each point using a Poisson model. A position was considered significantly enriched if the number of IP read counts was significantly higher (Z-score > 3) than the number of input read counts, after adjusting for the total number of reads sequenced in IP and input measurements. For each replicate set, if replicates were consistent (>80% agreement for top 40% of peaks/enrichment clusters), reads in each replicates were combined and enrichment regions were called as described above. Processed data are obtained using following parameters: genome version is FlyBase r5
Submission date Aug 02, 2013
Last update date May 15, 2019
Contact name DCC modENCODE
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
Platform ID GPL9058
Series (1)
GSE49488 H3K27ac (Active Motif).L3.Solexa
BioSample SAMN02299383
SRA SRX331369

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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