title: GSM3140926: Long-library_GFP- rep2; Drosophila melanogaster; OTHER
library_construction_protocol: GFP+ cells were mixed with 3x10^6 un-transfected S2 cells to increase the mRNA recovery of GFP+ cells. Total RNA was isolated using Qiagen RNeasy Maxi or Mini prep kits for GFP-and positive cells, respectively. PolyA+ RNA purification was performed using Invitrogen Dynabeads Oligo-dT25 (cat. no. 610-05) followed by Ambion TurboDNase (cat. no. AM2239) treatment according to manufacturers' protocols, also described previously in Arnold et al. Science 2013. TurboDNAse treated RNA was cleaned up using Qiagen RNeasy MinElute reaction clean up kit (cat. no. 74204) according to the manufacture's protocol. First strand cDNA synthesis was performed with 1µl of Invitrogen's SuperscriptIII (50°C for 60 min, 70°C for 15min; cat. no. 18080085) using a reporter-RNA specific primer (agttccttggcacccgagaattccaNNNNNNNNNNCGTGTGCTCTTCCGATCT) for 1-5µg of polyA+ RNA in 20µl total volume. We amplified the total amount of Gal4-DBD-candidate cDNA obtained from reverse transcription for Illumina sequencing by a 2-step nested PCR strategy using the KAPA Hifi Hot Start Ready Mix (KAPA Biosystems; cat. no. KK2602). In the first PCR (98°C for 45s; followed by 15 cycles of 98°C for 15s, 65°C for 30s, 72°C for 70s), cDNA was amplified using 2 Gal4-candidate-specific primers (AAGCCACCATGGAAAAG*G*C*C*A*T & AGTTCCTTGGCACCCGAGAA*T*T*C), one of which spans the splice junction of the mhc16 intron (5 and 3 nucleotides at the 3'ends are protected by phosphorothioate bonds, respectively), in a total of 10 reactions. This specifically amplifies the Gal4-DBD-candidate cDNAs and suppresses residual plasmid background. PCR products were purified by Agencourt AMPureXP DNA beads (ratio beads/PCR 0.9) and eluted in 20µl EB. The entire purified PCR product from each reaction served as template for the second PCR (98°C for 45s; followed by 8-12 cycles of 98°C for 15s, 65°C for 30s, 72°C for 15s) with the KAPA Hifi Hot Start Ready Mix using the following primers: aatgatacggcgaccaccgagatctacacXXXXXXXXacactctttccctacacgacgctcttccgatct (XXXXXXXX indicates the position of the index sequence for NGS)  and caagcagaagacggcatacgagatGTCGTGATgtgactggagttccttggcacccgagaattcca, which adds the overhangs that are required for flow cell hybridization and cluster generation prior to Illumina sequencing. PCR products were purified by Agencourt AMPureXP DNA beads (ratio beads/PCR 1.4), pooled and subjected to NGS (Illumina MiSeq platform).
attributes.tag: GEO Accession
attributes.value: GSM3140926