GSM7295712: UE_UAS_STARR-seq, CtBP, rep2; Drosophila melanogaster; OTHER
Total RNA was isolated using the Qiagen Rneasy Mini Prep Kit. mRNAs were enriched with Dynabeads Oligo-dT25 (Invitrogen), followed by a DNA digest with Turbo DNase (Ambion). mRNAs were cleaned up with AMPure XP DNA beads (Agencourt; ratio sample/beads 1:1.8) and then reverse transcribed with Superscript III (50°C for 60 min, 70°C for 15 min; Invitrogen 18080085) and a primer binding within the poly-A site of candidate mRNAs (reverse_transcription_rv: CTCATCAATGTATCTTATCATGTCTG). Next, RNA was digested with Rnase A (Thermo) for 1 h at 37°C, followed by bead cleanup of the remaining DNA (ratio sample/beads 1:1.4). All subsequent PCR reactions were prepared using the KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2602). A junction PCR was performed with a primer binding only in correctly spliced candidate mRNAs junction_primer_fw: AAGCCACCATGGAAAAG*G*C*C*A*T, junction_primer_rv: TATCATGTCTGCTCGAAG*C*G*G (98°C for 45s, (98°C for 15s,  65°C for 30 s, 72°C for 45 s)X15, followed by bead cleanup (ratio sample/beads 1:0.9). Sequencing ready PCR: candidate fragments were amplified (98°C 45 s, followed by 6-15 cycles, 98°C 15 s, 65°C 30 s, 72°C 30s) with the following primers: i5: aatgatacggcgaccaccgagatctacacXXXXXXXXacactctttccctacacgacgctcttccgatct (XXXXXXXX indicates the position of the index sequence for NGS) and the reverse primer TruSeq IDX with approptiate barcode for demultiplexing individual samples. PCR products were purified by Agencourt AMPure XP DNA beads (ratio sample/beads 1:0.8), pooled, and subjected to NGS. After another beads cleanup we measured each sample and made and equimolar mixture that was subjected to sequencing