GSM7917330: In vitro miCLIP2, unmethylated, rep 1; Homo sapiens; RIP-Seq
Each in vitro transcribed RNA in the library includes a transcript region of interest (146 nt), a unique molecular identifier (UMI, 15 nt) and the L3 linker sequence (21 nt). Transcript regions were selected based on miCLIP2 data from HEK293T cells to contain miCLIP2-detected m6A sites or unmodified adenosines in a DRACN context between positions 62–72 from the oligonucleotide start. In total, we selected 1,988 m6A sites in a DRACN motif ("m6A"), 1,500 unmodified adenosines in a DRACH motif ("Unmodified DRACH") and 400 permutated sequences ("Permutation"), where weak and strong DRACH motifs were exchanged within their original context. Each transcript sequence was represented by three different oligonucleotides with distinct UMIs (technical replicates). Differential methylation of the in vitro transcribed RNA libraries upon incubation with the recombinant METTL3-METTL14 writer complex or control was estimated using the published miCLIP2 protocol (PMID: 34157120), adjusted to the use of the in vitro transcribed RNA. Briefly, 500 ng purified RNA were used as input. Since fragment size was <200 nt and the L3 linker sequence was already included in the designed oligonucleotides, the initial RNA fragmentation and L3 linker ligation steps of the protocol were omitted. RNA crosslinking and immunoprecipitation with m6A-specific antibody, protein-RNA complex purification, proteinase K digestion, RNA isolation and reverse transcription were performed according to the miCLIP2 protocol. Truncations at the m6A-specific antibody crosslink are used to map the m6A position in the transcript. Library generation was carried out according to the published miCLIP2 protocol (PMID: 34157120). Equimolarly pooled in vitro methylation miCLIP2 libraries were sequenced on an Illumina NextSeq 500 sequencing machine as 141 nt and 25 nt paired-end reads including a 15 nt sample barcode.