title: GSM1193875: GFP_RNAi_IN_AB_(20130308_55_1_7); 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°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'), YL-1 (DRSC02765) 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 75cm^2 flasks.  30#g 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 10mL RT PBS, and centrifuged 2' at 600 x g.  Cells were resuspended in 4mL 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 degrees C and washed with 2mL HM2 and centrifuged 5' at 100 x g at 4 degrees C.  Nuclei were brought up in 450 microliters HM2, warmed to 37 degrees C for 2', CaCl2 was added to 1mM, and digested with 0.5U MNase for 10'. Following MNase digestion and termination with 5mM EDTA, nuclei were incubated for 30' on ice and then pelleted 5' at 400 x g at 4 degrees C and supernatant was saved on ice (S1).  Nuclei pellets were brought up in 600ul HE80 (10mM Hepes 7.4, 0.25mM EDTA, 80mM NaCl, 0.5mM PMSF) 15' on ice, and passed 4x through a 20ga needle and 4x through a 26ga needle to solubilize nucleosomes.  The soluble fraction was combined with S1 and clarified 2' at max speed.  An aliquot was set aside as input and 10 microliters H2Av antibody (Active Motif #39715) was incubated overnight on nutator.  Equlibrated Protein G Dynabeads were added for 3 hours to recover antibody and bound nucleosomes.  The beads were then washed at least 3x5' each with HE80.  DNA libraries were prepared using a protocol similar to PMID: 22025700 with modifications. We used the TruSeq oligo design to enable barcoding of samples 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: GSM1193875