Nebnext rrna depletion kit

Total RNA was isolated from the nasopharyngeal samples using Trizol LS reagent (ThermoFisher Scientific, Waltham, MA) in combination with the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA). RNA quantity was measured with the Qubit 2.0 fluorometer (ThermoFisher Scientific, Waltham, MA); its quality was assessed with the Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA) using the RNA 6000 Nano kit. Total RNA underwent DNase treatment using the TURBO DNA-free™ Kit (ThermoFisher Scientific, Waltham, MA) and rRNA reduction for both human and bacterial rRNA using NEBNext rRNA Depletion Kits (New England Biolabs, Ipswich, MA). RNA was prepared for sequencing using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA) and sequenced on an Illumina NovaSeq6000 using a S4 100PE Flowcell (Illumina, San Diego, CA). All RNAseq samples had sufficient sequence depth (mean, 8,067,019 pair-end reads/sample) to obtain a high degree of sequence coverage.

Total RNA was extracted from six pairs of freshly frozen LUAD tissues and matched controls, following treatment with NEB Next rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) to remove ribosomal RNA (rRNA) for the degradation of linear RNAs and enrichment of circRNAs. RNA libraries were constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) according to the manufacturer’s instructions. Subsequent transcriptome on an Illumina HiSeq 4000 was conducted by the Cloud-Seq Biotech Ltd. Co. (Shanghai, China). Significant differential expressed circRNAs were screened by fold change >2 or <−2 and p value < 0.05.

To investigate whether methylated modifications affect gene expression and their molecular mechanisms, we performed transcriptome sequencing for six samples and detected mRNAs, lncRNAs and circRNAs. Briefly, RNA was extracted using the TRIzol method, rRNAs were removed from total RNA using the NEBNext^® rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer’s instructions, and then, RNA sequencing libraries were constructed using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) after quality control. Library quality control and quantification were performed using the BioAnalyzer 2100 system (Agilent Technologies, USA). Subsequent sequencing of 150 bp paired-end reads on the Illumina Novaseq 6000 sequencer. Raw data was quality controlled by Q30, and the Cutadapt program was used to remove adaptors and low-quality reads. The high-quality reads were aligned to the chicken reference genome (GCF_000002315.6_GRCg6a), and then mRNA, lncRNA and circRNA were detected and identified. Subsequently, differential expression analysis and functional enrichment analysis were performed.

The total RNA of cells was extracted with RNAiso Plus (9109, Takara). RNA-seq was performed by Cloudseq Biotech Inc. (Shanghai, China) following the published procedure (25 (link)). The detailed information is listed below. Total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer’s instructions. RNA libraries were constructed by using NEBNext^® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150-bp paired-end reads.

Purified biotinylated transcripts were depleted of mature ribosomal RNAs using a NEBNext® rRNA Depletion Kit (New England Biolabs, cat# E6350), and used to prepare stranded sequencing libraries with a NEBNext Ultra II Directional RNA library Prep kit for Illumina and barcoded primers (New England Biolabs, cat# E7765) according to the manufacturer’s instruction. The barcoded libraries were quantified using NGSBIO Library Quant Kit Blue for Illumina® (PCR Biosystems, cat# PB71.15-01) and pooled prior to sequencing. Single-read sequencing was performed at the Oxford Genomics Centre, UK using a NextSeq 500 platform (Illumina, NextSeq 500/550 v2.5 Kits, 75 cycles) at ∼20 million reads per demultiplexed sample per single lane of the NextSeq 500 flow cell.

Transgene expression in HEK293 T-Rex Flp-In DHX36-KO DHX36 rescue cells and HEK293 T-Rex Flp-In DHX36-KO DHX36-E335A rescue cells was induced by addition of 500 ng ml⁻¹ tetracycline (Merck). In triplicates, RNA from the induced before-mentioned cell lines, wild-type HEK293 T-Rex Flp-In cells, and HEK293 T-Rex Flp-In DHX36-KO cells was isolated with TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. Depletion of ribosomal RNA was accomplished by using the NEBNext rRNA Depletion Kit (New England Biolabs).

Total RNA was extracted from mouse lungs using the TRIzol^® reagent (Thermo Fisher Scientific) according to the instructions of the manufacturer (n = 4–5, each group). The concentration and purity of the RNA samples were determined by automated optical density evaluation (OD₂₆₀/OD₂₈₀ ≥ 1.8 and OD₂₆₀/OD₂₃₀ ≥ 1.8) using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing (RNA-seq) libraries were prepared using a NEBNext rRNA Depletion Kit (New England Biolabs, Ipswich, MA, USA) and an ENBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) according to the instructions of the manufacturer using 500 ng of the total RNA samples. Next, 2 × 36 base paired-end sequencing was performed using a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) by Tsukuba i-Laboratory LLP (Tsukuba, Japan). Sequences were mapped to the mm10 mouse genome and quantified using CLC Genomics Workbench version 10.1.1 (QIAGEN). An adjusted P-value <0.01 (Benjamini–Hochberg FDR method for multiple testing corrections) and relative changes in transcription levels >1.2-fold were used as the cutoff criterion (Supplemental Figure 1). The data are available under GEO series accession number GSE181966.