GSM8265306: SUP-T1, RIP, rep2; Homo sapiens; RIP-Seq
1×107 cells/IP were resuspended in 1X ChIP Sonication Cell Lysis Buffer (Cell Signaling #56383). After centrifugation at 500 g for 3 min, Pellet (nuclear fraction) were suspended in 1X ChIP Sonication Nuclear Lysis Buffer (Cell Signaling #56383). Cell suspension were sonicated by Qsonica Q800R3 sonicator (Qsonica L.L.C, Newtown, CT) for 20 min. Chromatin was clarified (16,000× g, 4°C, 10 min) and incubated with DNase at 37°C for 20 min. 10 μg eIF4e and IgG antibody were incubated with chromatin overnight and then incubated with 30 µL Dynabeads Protein G (Invitrogen #10004D) for 4 hours and washed with low-salt washing buffer five times and high-salt washing buffer once. Elution and de-crosslinking were performed overnight in 150 µL elution buffer (300 mM NaCl, 5 mM DTT and 1% SDS in TE buffer, pH8.0) at 65 °C. RNA was extracted from chromatin by Trizol (ThermoFisher, #15596018) and processed according to the manufacturer's instructions. RNA was assessed for quality using the TapeStation (Agilent, Santa Clara, CA). Samples were prepared using the NEBNext rRNA Depletion Kit (NEB, E6310) and xGen Broad-range RNA Library Prep (IDT, 1009813) with xGen Normalase UDI Primers (IDT, various). Final libraries were checked for quality and quantity by TapeStation (Agilent) and Qubit (Thermo Fisher Scientific). This pool was subjected to 151bp paired-end sequencing according to the manufacturer's protocol (Illumina NovaSeq). Bcl2fastq2 Conversion Software (Illumina) was used to generate de-multiplexed Fastq files.