GSM3379587: MB185_rep1; Drosophila melanogaster; RNA-Seq
Fly heads were first isolated with a customized sieve. 400 frozen heads were added to 6 mL of 20 mM sodium acetate pH 8.5, 2.5 mM MgCl2, 250 mM sucrose, 0.5% NP-40, 0.6 mM spermidine, 0.2 mM spermine, 1 mM DTT, 1× complete protease inhibitor, 0.5 mg/mL torula RNA, 0.6 mg/mL carboxyl coated Dynabeads and 1.6 mg anti-GFP antibody. Homogenization was carried out on ice by 50 tractions in a Dounce homogenizer using the tight pestle followed by filtration over either a 10 or 20 μm cup filter (Partec: 0400422314, 040042315). Released chromatin and broken nuclei were adsorbed to carboxyl coated magnetic beads for 30 minutes at 4°C with constant rotation. Unbound antibody was removed by incubating the sample on ice for 20 minutes with 100 mL of washed UNOsphere SUPra resin (Bio-Rad: 1560218). After the resin was removed on a 10 μm cup filter and the carboxyl beads on a magnet stand, the nuclei-containing supernatant was mixed with an equal volume of 500 mM sodium acetate pH 8.5, 250 mM sucrose, 6 mM EGTA, 6 mM EDTA, 0.6 mM spermidine, 0.2 mM spermidine, 1 mM DTT, 1× complete protease inhibitor, 0.25 mg/mL torula RNA and 30 mL Protein A Dynabeads (ThermoFisher: 10002D). A 2-hour incubation on ice with occasional agitation was used to recover tagged nuclei. Bead-bound nuclei were then recovered on a magnet stand and washed twice with 250 mM sodium acetate pH 8.5, 250 mM sucrose and 0.1% NP-40. Nuclei were then released at 37°C for 1 hour by incubation in 50 μL of 10 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 250 mM sucrose, 0.1% NP-40, 1 mg/mL torula RNA, 40 units RNAsin (Promega: N2515), 2 units DNAseI (NEB: M0303L), 320 units IdeZ protease (NEB: P0770S). The sample was diluted to 100 μL with 10 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 250 mM sucrose and 0.1% NP-40, EGTA was added to 1 mM and the suspension was rapidly triturated 100 times. After returning the sample to a magnet stand, 90 μL of buffer containing released nuclei was removed and added to 1.5 μL of Protein G Dynabeads that were previously resuspended in 10 μL of 10 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 250 mM sucrose and 0.1% NP-40. The second binding reaction was run for 1 – 3 hours on ice with occasional agitation, followed by 2× 250 μL washes in 10 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 250 mM sucrose and 0.1% NP-40. Prior to the last wash a 5 μL aliquot was removed for quantitation and the remainder of the sample was solubilized in Arcturus Picopure RNA extraction buffer. Nuclear RNA was DNAseI treated and purified using the Arcturus PicoPure system exactly as instructed by the supplier. Purified RNA was mixed with a 1:100,000 dilution of ERCC standard RNA (ThermoFisher: 4456740) and amplified using the Nugen Ovation v2 system (Nugen: 7102-32). cDNA was then blunted, ligated to barcoded linkers (Nugen: 0319-32, 0320-32) and sequenced in paired-end mode on an Illumina HiSeq2500 to 125 nt read lengths.
GEO Accession
GSM3379587