RIP-Seq of HEK293T cells: HA-DDX3-2
RNA samples from RIP assay were prepared as described above. Next generation sequencing library preparations were constructed following the manufacturers protocol. For each sample, ChIP product was used for library preparation. The ChIP product was treated with End Prep Enzyme Mix for end repairing, 5 Phosphorylation and dA-tailing in one reaction, followed by ligation to adaptors with a T base overhang. Adaptor ligated DNA was then recovered using Beads. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 3.0 Fluorometer. Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq/Novaseq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA) or a MGI2000 instrument according to manufacturers instructions (MGI, Shenzhen, China). Sequencing was carried out using a 2x150 paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument; image analysis and base calling were conducted by the NovaSeq Control Software (NCS) + OLB + GAPipeline-1.6 (Illumina) on the NovaSeq instrument; image analysis and base calling were conducted by the Zebeacall on the MGI2000 instrument.