title: GSM5017077: Boar fresh sperm-IP, rep3; Sus scrofa; RIP-Seq
library_construction_protocol: The total RNA of fresh and frozen-thawed sperm was extracted using the TRIzol LS Reagent kit (Invitrogen, Carlsbad, CA, USA). The Ribo-Zero rRNA Removal Kit (Illumina, Inc., San Diego, CA, USA) was used to reduce the ribosomal RNA content of total RNA. Then, the RNA was chemically fragmented into about 100 nucleotides in length using fragmentation buffer (Illumina, Inc., CA, USA). RNA fragments were incubated with anti-m6A polyclonal antibody (Synaptic Systems, Göttingen, Germany) in immunoprecipitation (IP) buffer for 2 h at 4 °C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher Scientiﬁc, Waltham, MA, USA) for 2 h at 4 °C. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, Ann Arbor, MI, USA) in IP buffer and then extracted with Trizol Reagent. The NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) was used to construct RNA sequencing libraries for the non-immunoprecipitated input RNA samples (mRNA-seq) and immunoprecipitated IP RNA samples (MeRIP-seq). Libraries quality control was performed by BioAnalyzer 2100 analyzer (Agilent, Santa Clara, CA, USA) and high-throughput sequencing was subjected to 150-bp double-ended mode sequencing on Illumina HiSeq 4000 sequencer (Illumina, Inc., San Diego, CA, USA). The image analysis, base recognition, quality control and original reads (Raw Data) were generated after Illumina HiSeq 4000 sequencer sequencing.