m6A-seq and RNA-seq of 4 pairs NPC tissues with or without metastasis

Nasopharyngeal carcinoma (NPC), which arises from the nasopharyngeal pharynx, is a metastasis-prone malignancy highly prevalent in East and Southeast Asia, especially southern China. Despite progress in chemo-radiotherapeutic strategies, about 20% of NPC patients still suffer from distant metastasis, thus developing new therapeutic strategies for NPC is of vital importance. N6-methyladenosine (m6A) RNA modification, the most abundant epitranscriptomic modification in eukaryotic mRNA, has attracted great attention in cancer research because of its vital biological functions. However, little is known about the underlying molecular mechanisms between m6A modification and NPC progression. We aimed to identified differential m6A peaks by analyzing the m6A modification profile in NPC with paired tissues with or without metastasis using m6A-seq. For RNA-seq and ChIP-seq, we aimed to identified the downstream targets of CBX1 in NPC. Total RNA from 4 pairs NPC tissues with or without metastasis were extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quality and quantity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately more than 25 ug of total RNA representing a specific adipose type was used to deplete ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA). Following purification, the ribosomal-depleted RNA is fragmented into ~100-nt-long oligonucleotides using divalent cations under elevated temperature. Then the cleaved RNA fragments were subjected to incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) supplemented with BSA (0.5 μg μl−1). The mixture was then incubated with protein-A beads and eluted with elution buffer (1 × IP buffer and 6.7mM m6A). Eluted RNA was precipitated by 75% ethanol. Eluted m6A-containing fragments (IP) and untreated input control fragments are converted to final cDNA library in accordance with a strand-specific library preparation by dUTP method. The average insert size for the paired-end libraries was ~100±50 bp. And then we performed the paired-end 2×150bp sequencing on an Illumina Novaseq 6000 platform at the LC-BIO Bio-tech ltd (Hangzhou, China) following the vendor's recommended protocol. m6A seq and RNA seq of 4 pairs NPC tissues with or without metastasis