Poly t oligo attached magnetic beads

Total RNA was isolated using TRIzol reagent (Thermo Scientific), following by RNA purification by RNeasy MiniElute Cleanup Kit (Qiagen), according to the manufacturer’s instructions. For library construction, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Illumina) (Fig 1). The purified mRNA was cut into fragments with an average length of 155 bp using divalent cations at 95°C for 8 min. The cleaved mRNA fragments were reverse-transcribed into first strand of cDNA using random hexamers, prior to the synthesis of the second strand of cDNA. After end repair process, cDNA fragments were linked with adaptors and amplified by PCR.

After validating the integrity and purity of total RNA, cDNA library was constructed according to the protocol of TruSeq RNA Sample Prep Preparation Kit (RS-930-2001; Illumina, San Diego, CA). Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads (Illumina, San Diego, CA) from total RNA, and sheared to small fragments using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were subjected to end-repair using 3ʹ–>5ʹ exonuclease and polymerase, then go through the addition of a single ‘A’ base and subsequent adapter-ligation. The unsuitable fragments were removed using AMPureXP beads (Beckman Coulter Inc, Brae, CA, USA), and the sequencing library was constructed with PCR amplification. After quantified using the Quant-iT PicoGreen ds DNA Assay Kit (Invitrogen, Eugene, OR, USA) and fluorospectrophotometry and quantified with Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany), the multiplexed DNA libraries were mixed by equal volume with normalized 10 nM concentration. Finally, the sequencing library was then sequenced on Illumina Hiseq2000 platform (Shanghai Personal Biotechnology Cp., Ltd. Shanghai, China).

Total RNA was isolated from mLN‐ and pLN‐iFRCs using the AllPrep DNA/RNA kit (Qiagen). Agilent Technologies 2100 Bioanalyzer was used to confirm quality and integrity of total RNA. Poly A‐containing mRNA was purified via poly T oligo‐attached magnetic beads (Illumina). The mRNA was subsequently subjected to library preparation using Script Seq v2 Library preparation kit (Illumina). Deep sequencing was performed on Illumina HiSeq2500 using 50 bp single reads. The FastQC tool was used to assess sequenced libraries for read quality, which were then aligned versus the mouse reference genome (assembly: GRCm38) by using splice junction mapper Tophat2 v1.2.0 59 with default parameterization. The htseq‐count program was utilized to quantify reads aligned to annotated genes 60. Determined read counts served as input to DESeq2 61 for pairwise detection and quantification of differential gene expression. Only genes with annotated official Gene Symbol were included into heatmaps. From the raw read counts RPKM (reads per kilobase maximal transcript length per million mapped reads) values were computed for each library. The Gene Expression Omnibus (GEO) accession number for RNA‐seq data reported in this paper is GSE93670.

Libraries for sequencing were prepared in accordance with the Illumina TruSeq RNA sample preparation v2 guide (Part # 15026495, rev.D, September 2012) for Illumina Paired-End Indexed Sequencing. PolyA mRNA first underwent two rounds of purification using Illumina poly-T oligo-attached magnetic beads. During the second elution, the polyA mRNA was fragmented and primed with random hexamers for cDNA synthesis. After first strand cDNA synthesis, the RNA template was removed and a replacement strand was synthesized to generate double stranded cDNA. Ends were then repaired, dA base added and Illumina indexing adapters ligated. cDNA fragments with adapters on both ends underwent 15 cycles of PCR. Libraries were validated with the Agilent 2100 bioanalyzer to check size distribution. Samples were quantified by qRT-PCR, the concentrations normalized and samples pooled according to biological replicate. Pools were loaded at 10 pM onto four lanes of an Illumina flowcell v3 and sequenced using the Illumina HiSeq 1500, 2 X 100 bp paired-end run. Sequences are deposited in the NCBI Sequence Read Archive under the BioProject ID PRJNA385903.

Total RNA extracted from fibroblasts (treated with 0 or 1.0 mg/ml COS for 24, 48, and 72 h) was subjected to library construction and RNA Sequencing (RNA-seq) analysis after selection by RNA Purification Beads (Illumina, San Diego, CA, United States). Briefly, mRNA was purified from the total RNA of fibroblasts with poly-T oligo-attached magnetic beads (Illumina, San Diego, CA, United States). Then, the RNA was used as a template to synthesize the first strand of cDNA, and the first strand of cDNA was used as a template to synthesize the second strand of cDNA. The remaining protruding ends were converted into flat ends by exonuclease/polymerase activities followed by enzyme removal, and six RNA sequence libraries were constructed. Library preparation and sequencing were performed on an Illumina NovaSeq platform by Personal Biotechnology Cp., Ltd. (Shanghai, China). Raw data has been submitted to the Sequence Read Archive (SRA) database at the National Center for Biotechnology Information (NCBI) with the accession ID PRJNA860830, and Gene function was annotated based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The criteria for differentially expressed genes (DEGs) need to be greater than or less than a twofold change from the control.

A total of 3 μg RNA per sample was used for sequencing library construction. Libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions. Briefly, poly (A)-containing mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Illumina, San Diego, CA, USA). Then, the mRNA was broken into short fragments by a fragmentation buffer (Ambion, Austin, TX, USA); the fragments were used as templates for cDNA synthesis. First-strand cDNA was synthesized using random hexamer-primers and M-MuLV reverse transcriptase (RNase H). Second strand cDNA synthesis was performed using DNA Polymerase I (New England Biolabs) and RNase H (Invitrogen). Poly(A) sequences were added to the 3′ ends of cDNA fragments and sequencing adaptors with hairpin loop structure were ligated to the cDNA ends. Suitable fragments (150–200 bp) were selected by agarose gel purification and enriched by PCR amplification. Finally, the PCR amplicons were purified using magnetic beads (Illumina) and dissolved in EB solution to generate the sequencing libraries. Library quality was assessed on the Agilent 2100 Bioanalyzer.

Total RNA was isolated from the roots, stems, and leaves of five plants of the rice bean variety ‘Jingfan No. 1’ using Trizol Reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA was then treated with RNase-free DNase I (Takara, Otsu, Shiga, Japan) at 37°C for 30 min to remove residual DNA. The RNA quality was verified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and confirmed using RNase-free agarose gel electrophoresis. The concentration of the total RNA was further quantified using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, Delaware USA). Equal amounts of total RNA from rice bean ‘Jingfan No. 1’ were quickly frozen in liquid nitrogen for storage at -80°C until further use.
A cDNA library of pooled RNA was obtained using a TruSeq RNA Sample preparation kit (Illumina, USA), according to the manufacturer's instructions. Poly-T oligo-attached magnetic beads (Illumina Inc., San Diego USA) were used to isolate poly-A mRNA from total RNA. First-strand cDNA was synthesized from the fragmented mRNA using random hexamer primers and reverse transcriptase (Invitrogen, USA). The single-end cDNA library was prepared according to Illumina’s recommended protocol.