title: synthetic human 5HT2CR RNA amplification, inosine at A site; TGIRT enzyme; cyanoethylated
design: We ligated 10 pmol adaptor (5'-rApp-NNNNNNGATCGTCGGACTGTAGAA-3'SpC3, IDT) to the 3 of RNA with 200 U T4 RNA ligase 2, truncated KQ (NEB) in 1X T4 RNA Ligase Buffer supplemented with 20% (w/v) PEG8000 at 16 C overnight. We removed excess adaptors with 1.4X SPRI, and then reverse transcribed the RNA using a primer complementary to the 3 adaptor (5'-TTCTACAGTCCGACGATC-3', IDT). We followed the MaP protocol RT conditions, including 6 mM Mn2+ and 200 U SuperScript II (Thermo Fisher Scientific) at 42 C for 3 h to induce read-through and mutation at chemically-modified bases. We also included a template switch oligo in this reaction, designed with an ACA spacer42,43 (5'-CCTTGGCACCCGAGAATTCCAACAGGGGG-3'SpC3, IDT).  We PCR-amplified ligated- and template switched-products for 1619 cycles with indexing primers from the TruSeq Small RNA Library Prep Kit (RS-200-20012, 0024, 0036). We pooled each library and sequenced using 2 x 150 bp paired-end reads from the Illumina MiSeq Reagent Kit V2.