GSM1193877: H2Av_RNAi_3NT_AB_(20120921_34_5_5); Drosophila melanogaster; OTHER
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'), 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 150cm^2 flasks.  30 microg 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.  We modified protocols from  (PMID: 20007866; PMID: 22343941, 2012; PMID: 11340173) to fractionate cells and isolate nascent RNA.  dsRNA treated cells were harvested and pelleted by centrifugation for 1' at 1,000 x g at 4 degrees C.  Cells were quickly washed with 2mL ice cold PBS supplemented with 10 microg/mL alpha-Amanitin to inhibit transcription, split into two separate 2ml tubes, and then pelleted by centrifugation 3' at 3,000 x g.  The cells were then lysed in 0.9mL ice cold Buffer A (1microl/mL SUPERase*In, protease inhibitor (Roche), 10mM Hepes 7.9, 10mM KCl, 3mM MgCl2, 0.34M sucrose, 10% glycerol, 1mM DTT, 0.1% Triton X-100) supplemented with 10microg/mL alpha-Amanitin, 8' on wet ice with intermittent mixing.  Nuclei were then pelleted as before and brought up in 0.9mL buffer B (1microl/mL SUPERase*In 9mM EDTA, 0.2mM EGTA, 1mM DTT, protease inhibitor (Roche), 0.1% Triton X-100) 15' on wet ice, and repeated 2x or until nuclei lyse.  The chromatin pellet was then brought up in 0.9mL buffer B+ (1microl/mL SUPERase*In, 20mM EDTA, 0.2mM EGTA, 2mM spermine, 5mM spermidine, 1mM DTT, protease inhibitor (Roche)) and washed 3x.  The final insoluble pellet was brought up in 1mL Buffer B+.  The two pellets from the same original flask were pooled and dissolved in 10mL Trizol and RNA was isolated according to manufacturer's instructions. Nascent RNA was treated with DNase (RQ1, Roche) and extracted with acid phenol and ethanol precipitated with GlycoBlue and 0.3M NaOAc.  We selected for the presence of a 5' cap using Terminator 5'-Phosphate-Dependent Exonuclease (Epicentre), as described (PMID: 20007866). Nascent, capped RNA was purified with acid phenol as before. Libraries were constructed as described for NET-seq (Churchman and Weissman, 2012) with the following modifications.  Ligated RNA was digested with pH 10.3 alkaline fragmentation buffer for 30'.  Reverse transcriptase primer concentration was reduced to 1.09 microM in RT reaction mix.  Libraries were amplified with KAPA HiFi under limiting cycles.  Following amplification, PCR reactions were purified with AMPure XP beads at a 1:1 ratio before PAGE purification to aid in the elimination of empty products.  Biological replicates were sequenced for each condition.
GEO Accession
GSM1193877