GSM7083144: H1, WT, IP, acRIP-Seq rep2; Homo sapiens; RIP-Seq
Cells from WT and NAT10 KD hESCs were collected and total RNA was harvested with Trizol reagent (Invitrogen, cat. NO 15596026). Remove the rRNAs from the total RNA by using the Ribo-Zero rRNA Removal Kits (Illumina, USA). The RNA was randomly fragmented into fragments of about 200 nt. Protein A/G magnetic beads (Thermo Fisher Scientific, MA, United States) and ac4C antibody (ab252215, Abcam, Cambridge, United Kingdom) or control rabbit IgG antibodies (GS-ET-005, GenSeq, Shanghai, China) were rotated and incubated at room temperature for 1 hour to bind the antibody to the magnetic beads. Then the RNA fragments and the antibody bound with magnetic beads were rotated and incubated at 4 ℃ for 4 hours. RNA/antibody complexes were eluted from magnetic beads by Protease K and RNA was extracted by phenol:chloroform method. RNA sequencing libraries were constructed using the NEBNext® Ultra II Directional RNA Library Prep Kit (Thermo Fisher Scientific, MA, United States). The constructed libraries were quality-controlled with Agilent 2100 bioanalyzer and then sequenced on Illumina NovaSeq 6000 sequencer (Illumina, Inc., San Diego, CA, United States) for high-throughput sequencing. ac4C-RIP-seq