title: GSM3265470: Tm3_d1_rep1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: INTACT protocol: Adult flies were entrained to a 12:12 light:dark cycle, anesthetized by CO2 at ZT8 - ZT12, flash frozen in liquid N2 and decapitated by vigorous vortexing. Heads or wings/appendages were then collected on cooled metal sieves (H&C Sieving Systems: 1296, 1297, 1298, 1301). Both flies and purified frozen material can be stored indefinitely at -80degrees C. In a typical experiment 100 to 500 frozen heads were added to 5mls of 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1X complete protease inhibitor (Sigma: 5056489001), 3mg/ml BSA (ThermoFisher: AM2618), 1mg/ml torula RNA (ThermoFisher: AM7118), 0.6mg/ml carboxyl coated Dynabeads (ThermoFisher: 14306D) and 2 microgram anti-GFP antibody (ThermoFisher: G10362). Homogenization was carried out on ice by 50 tractions in a Dounce homogenizer using the tight pestle followed by filtration over a 10 micrometer cup filter (Partec: 0400422314). Released chromatin and broken nuclei were adsorbed to carboxyl coated magnetic beads for 30 minutes at 4 degrees C with constant rotation. After which the beads were removed on a magnetic stand, the supernatant diluted to 50mls with 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT and 1X complete protease inhibitor (Sigma: 5056489001), filtered over a 1 micrometer cup filter (Pluriselect: 435000103) and split into two equal volumes. A 40% Optiprep (Sigma: D1556), 20mM Beta-glycerophosphate pH7, 2mM EDTA and 0.5% NP40 solution was then carefully placed under each aliquot. Followed by a lower layer of 50% Optiprep, 20mM Beta-glycerophosphate pH7, 2mM EDTA and 0.5% NP40. Nuclei were then pelleted on to the 50% layer for 30 minutes at 2300Xg. Purified nuclei were passed over a 10 micrometer cup filter, diluted to 10mls with 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT and 1X complete protease inhibitor and incubated with 30 microliters of protein G Dynabeads (ThermoFisher: 10004D) for 40 minutes on ice with occasional agitation. Bead-bound nuclei were recovered on a magnet stand followed by a 20 minute incubation on ice in 9mls of 20mM Beta-glycerophosphate pH7, 300mM NaCl, 1M urea, 0.5% NP40, 2mM EDTA, 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1X complete protease inhibitor, 0.075mg/ml torula RNA and 0.05Units/ml Superasin (ThermoFisher: AM2696). Nuclei were then recovered on a magnet stand, resuspended in 1ml of the previous buffer, passed over a 10 micrometer cup filter, a 5 microliter aliquot was withdrawn for quantitation and the remainder of the sample solubilized in Arcturus Picopure RNA extraction buffer (ThermoFisher: KIT0204). TAPIN-seq protocol: 100 to 3000 frozen heads were added to 5mls of sodium acetate ph8.5, 2.5mM MgCl2, 250mM sucrose, 0.5% NP-40, 0.6mM spermidine, 0.2mM spermine, 1mM DTT, 1X complete protease inhibitor, 0.5mg/ml torula RNA, 0.6mg/ml carboxyl coated Dynabeads and 2microgram 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 micrometer cup filter (Partec: 0400422314, 040042315). Released chromatin and broken nuclei were adsorbed to carboxyl coated magnetic beads for 30 minutes at 4 degrees C with constant rotation. Unbound antibody was removed by incubating the sample on ice for 20 minutes with 100 microliters of washed UNOsphere SUPra resin (Biorad: 1560218). After the resin was removed on a 10 micrometer cup filter and the carboxyl beads on a magnet stand, the nuclei-containing supernatant was mixed with an equal volume of 500mM sodium acetate ph8.5, 250mM sucrose, 6mM EGTA, 6mM EDTA, 0.6mM spermidine, 0.2mM spermidine, 1mM DTT, 1X complete protease inhibitor, 0.25mg/ml torula RNA and 30 microliters 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 250mM sodium acetate ph8.5, 250mM sucrose and 0.1% NP40. Nuclei were then released at 37 degrees C for 1 hour by incubation in 50 microliters of 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose, 0.1% NP40, 1mg/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 microliters with 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40, EGTA was added to 1mM and the suspension was rapidly triturated 100 times. After returning the sample to a magnet stand, 90 microliters of buffer containing released nuclei was removed and added to 1.5 microliters of Protein G Dynabeads that were previously resuspended in 10 microliters of 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40. The second binding reaction was run for 1 to 3 hours on ice with occasional agitation, followed by 2 X 250 microliters washes in 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40. Prior to the last wash a 5 microliter aliquot was removed for quantitation and the remainder of the sample was solubilized in Arcturus Picopure RNA extraction buffer.