METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection by Li et al.

Published: 9 April 2021| Version 1 | DOI: 10.17632/6n65z4sv9c.1
Contributor:
Na Li

Description

Table S1 SARS-CoV-2 Me-RIP-Seq Peaks Summary Table S2 SARS-CoV-2 Control METTL3 KD MeRIP-Seq Peaks Summary Table S3 shM3 Vs NTC RNA Seq Table S4 Primers used

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MeRIP-Seq and MeRIP-qPCR: Briefly, supernatant or cellular RNAs were purified from viral infected-Vero or Caco-2 cells. The RNAs were treated with DNaseI, concentrated and quantified. Purified RNA was fragmented to 100–200 nucleotides using Ambion RNA Fragmentation Reagent (AM8740, Life Technologies) and the fragmented RNA purified by ethanol precipitation. An input sample (10% of total fragmented RNA) was reserved. Fragmented RNA was incubated with 10 μl rabbit anti-m6A polyclonal Ab (ab151230, Abcam) in IP binding buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, pH 7.4) for 2 h at 4°C. The mixture was then incubated with 50 μl protein A/G magnetic beads (Thermo Fisher) for 2 h at 4°C, and the beads were collected and washed twice in IP wash buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% NP-40, pH 7.4). Bound RNA was eluted from the beads with m6A elution buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% NP-40, 25 mM m6A, pH 7.4) and extracted with RNA Clean & Concentrator Kits (ZYMO). Input and m6A-containing viral RNA were dissolved in water and either reverse transcribed by iScript cDNA synthesis kit (Bio-Rad) and analyzed by qPCR or processed for library generation using a TruSeq mRNA library preparation kit (Illumina). Sequencing was performed by Illumina NovaSeq 6000 at the IGM Genomics core, UCSD. MeRIP-Seq Data Analysis Sequencing reads were aligned to SARS-CoV-2 or human genome by Bowtie2 or STAR. Then m6A peaks were called by m6A viewer or MACS2 based on its paired m6A-RIP/input data from the aligned reads: m6A viewer (expected peak length 200, enrichment fold>3, FDR<0.01, Minimum Peak Height :10000) and MACS2 (p-value< 0.05, call-summit, no model, BAMPE, maximum duplicate fragments=1 or keep all duplicates for SARS-CoV-2, and keep-dup auto, extsize 200, nomodel, p-value<1e-5 for human). The peak region and summit of each alignment and peak calling strategy were summarized in Supplementary table 1 and 2. RNA-Seq and Data Analysis For RNA-Seq, total RNAs were extracted from SARS-CoV-2-infected shControl and shMETTL3 Caco-2 cells (three biological replicates). Sequencing was performed by Illumina NovaSeq 6000 at the IGM Genomics Center, UCSD. Fastqc was used to perform quality control on sequencing data, and Cutadapt was used to remove adapters and trim reads. The preprocessed reads were then aligned to the human or SARS-CoV-2 genome using STAR. The raw gene count for each sample was obtained by htseq-count and the differential gene expression analysis was performed by DEseq2.