GSM6285205: RDseq, zfh1, 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 second strand PCR was performed with a primer binding upstream of the intron sequence which is part of candidate mRNA (2nd_strand_primer_fw: TTGGTAAAGCCACCATGGAAAAG*G) (98°C for 60 s, 65°C for 30 s, 72°C for 90 s), followed by bead cleanup (ratio sample/beads 1:1.4). In the next step, unique molecular identifiers (UMIs) were introduced to the 3' ends of DNA fragments in a linear PCR with a primer binding to the Illumina i7 adaptor sequence (UMI_primer_rv: CAAGCAGAAGACGGCATACGAGATNNNNNNNNNNGTGACTGGAGTTCAGACGTGT*G) (98°C for 60 s, 65°C for 30 s, 72°C for 90 s). After bead cleanup (ratio sample/beads 1:1.4), the generated fragments were PCR-amplified (98°C 45 s, followed by 16 cycles, 98°C 15 s, 65°C 30 s, 72°C 70s) using two candidate-specific primers (junction_PCR_fw: AAGCCACCATGGAAAAG*G*C*C*A*T and junction_PCR_rv: CAAGCAGAAGACGGCATACG*A), one of which spans the splice junction of the mhc16 intron (5 and 1 nucleotides at the 3' ends are protected by phosphorothioate bonds, respectively). After another bead cleanup (ratio sample/beads 1:1), candidate fragments were amplified (98°C 45 s, followed by 6-15 cycles, 98°C 15 s, 65°C 30 s, 72°C 70s) with the following primers: i5: aatgatacggcgaccaccgagatctacacXXXXXXXXacactctttccctacacgacgctcttccgatct (XXXXXXXX indicates the position of the index sequence for NGS) and the reverse primer seq_ready_rv: CAAGCAGAAGACGGCATACGAGA*T. PCR products were purified by Agencourt AMPure XP DNA beads (ratio sample/beads 1:0.9), pooled, and subjected to NGS.