Truseq stranded total rna kit

Three biological replicates were used for RNA-seq analyses. The TruSeqTM Stranded Total RNA Kit (Illumina, San Diego, USA) and SuperScript double-stranded cDNA synthesis kit (Invitrogen, USA) were used to remove ribosomal RNA (rRNA) and synthesize cDNA. Illumina HiSeq X Ten sequencing platform was used to perform high-throughput sequencing. The sequencing read length was paired-end (PE) 150, and a 15G rRNA library was constructed.

The circRNA and mRNA library were constructed using the TruSeqTM Stranded Total RNA Kit (Illumina, San Diego, CA). And the ribosomal RNA (rRNA) was removed from 2 μg total RNA by the Ribo-Zero Magnetic kit (EpiCentre Biotechnologies, Madison, WI, USA). The RNA was fragmented and reverse-transformed to synthesize cDNA, then the adaptor was ligated. The cDNA second strand was digested with UNG enzyme, amplified by polymerase chain reaction (PCR), and purified to obtain the final library. The miRNA library was constructed using the TruSeqTM Small RNA sample prep Kit (Invitrogen) according to the instructions. After removing the rRNA, the 3' and 5' end adapters were connected to the kit separately, and then the primers were reversed and PCR cycles were performed. Next, the library was enriched, purified and quantified [27 ]. At last, high-throughput sequencing was conducted using the Illumina NovaSeq 6000 ( Supplementary Fig. 1).

The RNA-Seq transcriptome strand library was constructed according to the TruSeq^TM Stranded Total RNA kit from Illumina (San Diego, CA, USA), which was prepared with 5 μg of total RNA. The first cDNA strand was synthesized by using an IlluminaRibo-Zero Magnetic kit ribosomal RNA (rRNA) depletion with random hexamer primers. Then, the RNA template was removed, the replacement strand was synthesized, and dUTP was replaced with dTTP to generate ds cDNA. Using AMPure XP beads to separate ds cDNA from the reaction mixture. When a single “A” nucleotide was added to the 3′ end, multiple index adapters would also be connected to the ends of ds DNA. The cDNA size was selected on 2% UltraAgarose, and then PCR amplification was performed using Phusion DNA polymerase (NEB). After quantification by TBS380, the paired-end RNA sequencing library was sequenced with Illumina HiSeq 4000. In addition, 3 μg of total RNA was connected to the sequencing adapter using TruSeq^TM Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Subsequently, cDNA was synthesized by reverse transcription and a library was generated. Finally, using the library, we conducted deep sequencing at Shanghai Meiji Biomedical Biotechnology Co., Ltd. (Shanghai, China).

An amount of 5 μg of total RNA from subcutaneous adipose tissue samples was used to prepare RNA-seq transcriptome strand library using TruseqTM stranded total RNA kit (Illumina, San Diego, CA, USA). Ribosomal RNA depletion instead of poly A purification was performed with the Ribo-Zero Magnetic kit and then fragmented with fragmentation buffer. First-strand cDNA was generated with random hexamer primers. RNA templates were then eliminated to make a replacement strand, containing dUTP for the synthesis of double-stranded cDNA (dscDNA). AMPure XP beads were used to extract the dscDNA from the second strand reaction mix. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating together during the adapter ligation process and, eventually, multiple indexing adapters were ligated at the ends of the dscDNA. The sizes of libraries were selected for cDNA with a target fragment size of 200–300bp in 2% low range ultra-agarose, followed by PCR amplification using Phusion DNA polymerase (NEB, Ipswich, MA, USA) for 15 cycles. A pair-end RNA-seq sequencing library was sequenced with the Illumina Hiseqxten (2 × 150 bp read lengths) after TBS380 quantification.

Total RNA was extracted from mouse lung tissue using TRIzol ^® Reagent according to the manufacturer’s instructions (Invitrogen). Library construction and RNA sequencing were performed by Shanghai Majorbio Bio-Pharm Biotechnology Co., Ltd (Shanghai, China). Briefly, RNA-seq transcriptome strand library was prepared following TruSeq^TM stranded total RNA Kit from Illumina (San Diego, CA, United States) using 5 μg of total RNA. Shortly, ribosomal RNA (rRNA) depletion instead of poly(A) purification was performed by Ribo-Zero Magnetic kit and then fragmented by fragmentation buffer. Then double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, United States) with random hexamer primers, followed by the ligation of multiple indexing adapters to the ends of the double-stranded cDNA. After the libraries were quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq X ten (2 × 150 bp read length). All raw data for RNA sequencing were deposited into NCBI (GEO accession number GSE164963).

RNA was extracted from myocardial biopsies using Qiagen All-Prep DNA/RNA Kit (Qiagen, Venlo, The Netherlands), and libraries were prepared using Illumina TruSeq Stranded Total RNA Kit (Illumina, San Diego, CA, USA) according to the manufactures’ manuals. Sequencing was performed as paired-end sequencing, with a read length of 150 bps on the Illumina NovaSeq 6000 platform.

RNA was extracted from TRIzol-lysed PBMCs using the miRNAeasy Mini Kit (Qiagen). RNA quantity was measured on the Nano Drop 2000 Spectrophotometer (Thermo Scientific) (56.6 ± 16.7 ng μl⁻¹) and the quality and integrity measured with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All RNA integrity numbers (RINs) were greater than 6 (RIN: 7.66 ± 0.79). The Illumina TruSeq Stranded Total RNA kit (Ilumina, San Diego, CA, USA) was used for library preparation accordingly to manufacturer instructions without any modifications. A total of 80 indexed RNA libraries were pooled and sequenced using long read paired-end chemistry (2 × 150 bp) at a read depth of 30 M reads per sample using the Illumina HiSeq2500. Adapter sequences were clipped and low quality reads were discarded using Trimmomatic^{27 (link)} using parameters: ILLUMINACLIP, MINLEN:140, CROP:140. All high-quality trimmed reads were then mapped to UCSC Homo sapiens reference genome (build hg37) using default STAR v2.4.0 parameters²⁸ (percentage of mapped reads: 92.1% ± 1.6%). Samtools was used to convert bamfiles to samfiles and featureCounts^{29 (link)} was used to quantify gene expression levels for each individual sample using default paired-end parameters.