title: GSM1254261: Female_Ovary_Dam_rep1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Genomic DNA was extracted using Qiagen's DNEasy Blood and Tissue Kit components. Tissue was homogenized in 175 µl PBS with a plastic pestle. 2 µl of RNAse A (100 mg/ml) (Qiagen) were added and the homogenate was incubated for 2 minutes at room temperature. 20 µl of proteinase K (Qiagen) and Buffer AL were added and the mixture was incubated at 70 ˚C for 10 minutes. 200 µl of ethanol were added to the sample and it was transferred to a column and spun at 8000 rpm for 1 minute. The flowthrough was discarded and the column was transferred to a new collection tube. 500 µl of AW1 solution was added to the column and it was spun at 8000 rpm for 1 minute. The column was again transferred to a new collection tube and 500 µl AW2 solution was added and the column was spun at 13000 rpm for 3 minutes. The column was transferred to a new collection tube and 200 µl of AE solution was added and incubated on the column at room temperature for 30 minutes. The column was spun at 8000 rpm for 1 minute and the flowthrough containing the eluted genomic DNA was stored at 4 ˚C. A second elution was performed by adding an additional 200 µl of AE to the column, allowing it to incubate at room temperature for 10 minutes before spinning it at 8000 rpm for 1 minute. DamID Protocol: 300 ng of gonad genomic DNA were brought to a volume of 500 µl with ddH20.1 ml 100% ethanol and 50 µl 3M NaAcetate pH 5.2 were added. The solution was mixed gently, vortexed for 2 seconds and incubated at -20 ºC for 20 minutes. The tube was spun at 13 K RPM 30 minutes at 4 ºC. The pellet was washed with 500 µl of 70% ethanol and spun at 13K RPM for 10 minutes at 4 ºC. The remaining ethanol was removed and the pellet was dried. The precipitated DNA was digested with DpnI in a 50 µl volume containing 5 µl 10X buffer 4, 43.5 µl ddH2O and 1.5 µl DpnI (New England Biolabs, NEB). The reaction mixture was added to the dry pellet and used to gently resuspend the DNA by pipetting up and down several times. The DNA was digested overnight at 37 ºC. The following day the tubes were quickly spun (<5 seconds) to bring down condensation and the digest solution was transferred to a 0.2 ml tube and placed at 80 ºC for 20 minutes. Following incubation, the DNA was cleaned through Qiagen’s PCR Purification Qiaquick column following the product manual, eluted in 30 µl ddH20 and placed on ice.  The DamID adaptors (AdRt 5’-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3’ and AdRb  5’-TCCTCGGCCG-3’) were used to make a 50 µM stock solution of doublestranded adaptors by combining equal volumes of 100 µM singlestranded adaptors, heating the solution to 100 ºC for 1 minute and allowing the solution to cool slowly at room temperature. The doublestranded adaptors were ligated to the cleaned DpnI digested genomic DNA in a 20 µl reaction volume containing 15 µl of DpnI digested DNA, 2 µl 10X T4 ligation buffer (New England Biolabs), 0.8 µl of 50 µM doublestranded adaptors, 1.2 µl H20 and 1.0 µl T4 DNA ligase and incubated at 16  ºC for 2 hours, then 65 ºC for 10 minutes.  All 20 µl of the adaptor-ligated DNA were then subjected to DpnII digestion in a reaction solution composed of 8 µl 10X DpnII buffer (New England Biolabs), 51 µl H20, and 1 µl DpnII and incubated at 37 ºC for at least one hour. The DpnII digested DNA was PCR-amplified with a primer that recognizes the adaptor sequence (AdR_PCR Primer 5’-GGTCGCGGCCGAGGATC-3’). The PCR reaction mixture contained: 20 µl DpnII digested DNA, 8 µl 10X Taq Buffer with (NH4)2S04 but no MgCl2 (Fermentas), 1.25 µl 50 µM AdR_PCR Primer, 3.2 µl 25 mM MgCl2, 1.6 µl 10 mM dNTPs, 1.6 µl taq enzyme (Fermentas) and 44.35 µl H20.  The DNA was amplified using the following program: 10 minutes at 68 ºC, 1 minute at 94 ºC, 5 minutes at 65 ºC and 15 minutes at 68 ºC followed by 3 cycles of 1 minute at 94 ºC, 1 minute at 65 ºC and 10 minutes at 68 ºC and then 17 cycles of 1 minute at 94 ºC, 1 minute at 65 ºC and 2 minutes at 68 ºC.  3 µl were run on a 1 % agarose gel to check fragment size distribution which typically ranged between 400 and 2000 bp. Following amplification, the DNA was purified through a Qiagen Qiaquick column and eluted with 30 µl dd H20. Libraries were made using the ChIP-seq sample preparation kit (Illumina). Adapter-ligated DNA of 250-350 bp range was excised from the gel before PCR amplification.
attributes.tag: GEO Accession
attributes.value: GSM1254261