title: GSM1193880: H2Av_RNAi_MNase_AB_(20120606_77_6_4); Drosophila melanogaster; MNase-Seq
library_construction_protocol: Drosophila S2-DRSC cells were obtained from the Drosophila Genomics Resource Center (Stock #181) and only passages 10-20 were used for experiments.  Cells were cultured in Schneider's media supplemented with 10% heat inactivated FBS at 25 degrees C, with an ~30hr doubling time.  dsRNA was synthesized using NEB reagents and protocols from PCR templates containing the T7 promoter sequence.  PCR primers were as follows: H2Av Forward (5'- TAATACGACTCACTATAGGGCGAAACCGAATTCCGTAGAA - 3') H2Av Reverse (5- TAATACGACTCACTATAGGGAGTAGGCCTGCGACAGA -3'), and GFP control (PMID: 16204180). To administer dsRNA, 2.5 x 10^6  log phase cells / cm^2  surface area were brought up in Schneider's without FBS (0.1mL/cm^2) and seeded in 25cm^2 flasks.  30microg dsRNA/1x10^6 cells was incubated with cells for 1 hour with intermittent mixing.  After incubation with dsRNA, an equal volume of Schneider's with 20% FBS was added and cells were grown for 96 hours before harvesting.  Cells were pelleted 2' at 600 x g, washed with 5mL RT PBS, and centrifuged 2' at 600 x g.  Cells were resuspended in 1.9mL ice cold HM2 (10mM Hepes pH 7.4, 2mM MgCl2, 0.5mM PMSF), incubated 2' on ice, and lysed with 0.6% NP-40 with intermittent pipetting for 5' on ice.  Nuclei were centrifuged 10' at 100 x g at 4°C and washed with 1mL HM2 and centrifuged 5' at 100 x g at 4 degrees C.  Nuclei were brought up in 150microl HM2, warmed to 37 degrees C for 2', CaCl2 was added to 1mM, and digested with 0.066U MNase.  50 microl aliquots were taken out and terminated with 5mM EDTA on ice after 5', 10', and 15'.  Time-points that were similarly digested to mostly mononucleosomes (~80%) were sequenced using a protocol similar to PMID: 22025700 with modifications. We used the TruSeq oligo design to enable barcoding of libraries and modified AMPure XP to sample ratio to 1:1 to accommodate longer adapter sequences.  Biological replicates were sequenced for each condition.
attributes.tag: GEO Accession
attributes.value: GSM1193880