GSM1655082: Input_Male1; Drosophila melanogaster; RNA-Seq
Translated mRNA purification was performed as previously described (Thomas, A., P. J. Lee, J. E. Dalton, K. J. Nomie, L. Stoica et al., 2012 A versatile method for cell-specific profiling of translated mRNAs in Drosophila. PLoS One 7: e40276)  with the following modifications: approximately 1,000 heads per genotype were collected and homogenized in extraction buffer (20mM HEPES, pH 7.4, 150mM KCl, 5mM MgCl2, 0.5mM DTT, 100 g/mL Cyclohexamide, 100U/mL RNase OUT, 1X Complete Protease Inhibitor (Roche)). The sample was centrifuged at 4°C for 10 minutes at 2,000 x g and the supernatant was collected. 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC; Avanti®Polar Lipids, Inc.) and NP-40 were added to a final concentration of 30mM and 1%, respectively, to the supernatant and incubated on ice for 5 minutes. The lysate was then centrifuged at 4°C for 10 minutes at 20,000 x g. 40 μL of the supernatant was retained and placed directly into 200 μL of Lysis Solution  [Ambion® MicroPoly(A) Purist Kit] to be used to analyze the input RNA fraction. The remaining supernatant was added to Dynal Protein G magnetic beads conjugated to mouse anti-GFP (Memorial Sloan-Kettering Monoclonal Antibody Facility19C8/19F7). The lysate-bead slurry was incubated for 30 minutes at 4°C followed by washing in Wash Buffer (20mM HEPES, pH 7.4, 5mM MgCl2, 350mM KCl, 1% NP-40, 0.5mM DTT, and 100μg/mL Cyclohexamide) at 4°C. The IP was resuspended in Lysis Solution [Ambion®  MicroPoly(A) Purist Kit] and mRNA was directly isolated following the manufacturer’s protocol. Poly(A)+ transcripts were isolated using Ambion® MicroPoly(A)Purist™ Kit. 100 ng mRNA was chemically fragmented to a range of approximately 200-500 base pairs using the Ambion® RNA Fragmentation Reagent, and the reaction was cleaned using Zymo Research RNA Clean & Concentrator™-5. First strand cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen™) and a combination of 3 µg random hexamers and 0.15 µg oligo(dT)20 primers. Following first strand synthesis, the second strand of the cDNA was synthesized by addition of DNA polymerase I (Invitrogen™), RNase H (New England Biolabs® Inc.), dNTPs and second strand buffer (Invitrogen™).  The DNA from this and all subsequent reactions was purified using Zymo Research DNA Clean & Concentrator™-5 kit. Double stranded cDNA templates were blunt ended using  End-It™ Repair Kit (Epicentre®). Next, A-overhangs were added to both ends with Klenow fragment (3'→5' exo-minus) (New England Biolabs® Inc.). Illumina sequencing adapters were then ligated to both ends of the cDNA templates using Fast-Link™ DNA Ligation Kit (Epicentre®). cDNA templates were amplified by performing polymerase chain reaction (PCR; 18 cycles). The product was isolated by gel purification of 250-550 base pair fragments.
GEO Accession
GSM1655082