Nebnext multiplex small rna library prep kit

Additional high-throughput sequencing of zebrafish methylome was carried out using a modified m⁶A-seq method, which is similar to previously reported methods^{19 ,20 (link)}. Briefly, total RNA and mRNA were purified as previously described for m⁶A-seq. Purified mRNA (1 μg) was mixed with 2.5 μg of affinity purified anti-m⁶A polyclonal antibody (Synaptic Systems) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 hours at 4 °C. The mixture was subjected to UV-crosslinking in a clear flat-bottom 96-well plate (Nalgene) on ice at 254 nm with 0.15 J for 3 times. The mixture was then digested with 1 U/μl RNase T1 at 22 °C for 6 min followed by quenching on ice. Next, the mixture was immunoprecipitated by incubation with protein-A beads (Invitrogen) at 4 °C for 1 hr. After extensive washing, the mixture was digested again with 10 U/μl RNase T1 at 22 °C for 6 min followed by quenching on ice. After additional washing and on beads end-repair, the bound RNA fragments were eluted from the beads by proteinase K digestion twice at 55 °C for 20 and 10 min, respectively. The eluate was further purified using RNA clean and concentrator kit (Zymo Research). RNA was used for library generation with NEBNext multiplex small RNA library prep kit (NEB). Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions.