Next ultra directional rna library prep kit

We next aimed to characterise the host expression response to KV infection, and whether this differed between males and females. We injected 6 vials of 10 flies for each sex with either the control gradient solution or with KV. After 3 DPI, we homogenised flies in Trizol, extracted total nucleic acid, and enriched the sample for mRNA through DNAse treatment and poly-A selection. We used the NEB Next Ultra Directional RNA Library Prep Kit to make strand-specific paired-end libraries for each sample, following manufacturer’s instructions. Libraries were pooled and sequenced by Edinburgh Genomics (Edinburgh, UK) using three lanes of an Illumina HiSeq 4000 platform with strand-specific 75 nucleotide paired end reads. We subsequently identified a low level of Drosophila A Virus (DAV) contamination in both KV treated and untreated flies, reflecting the widespread occurrence of this virus in fly stocks and cell cultures. All reads have been submitted to the European Nucleotide Archive under project accession ERP023609.
We used SPAdes genome assembler (v3.11.1; [43 (link)]) to assemble the KV genome from RNA-sequencing reads, using the previously published genome (NC_033829.1) as an ‘untrusted contig’ (File S1).

Six libraries with two independent biological replicates were prepared using the Next Ultra Directional RNA Library Prep Kit (NEB) according to the manufacturer’s instructions. Briefly, mRNA was purified from total RNA using oligo dT attached to magnetic beads. Following purification, the mRNA was fragmented and first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (NEB). Subsequently, the cDNA fragments were blunt ended and linked with adaptors. Sequencing was performed using an Illumina HiSeq2500 platform at Novogene Bioinformatics Technology Co., Ltd (Beijing, China).

Total RNA was extracted from the second internodes by using RNAprep Pure Plant Kit (Tiangen, Beijing, China) and then treated with RNase-free DNase I (Takara) to remove genomic DNA. The RNA was electrophoresed on 1% agarose gels for monitoring RNA degradation and contamination. RNA purity, concentration, and integrity were further assessed using Nanodrop (Thermo Scientific, USA) and Agilent 2100 (Agilent Technologies, CA, USA). Twelve RNA-Seq libraries (4 time points × 3 independent biological replicates) were prepared using the Next Ultra Directional RNA Library Prep Kit (NEB), following manufacturer's instructions. In brief, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. The mRNA was fragmented into small pieces by using a divalent cation under elevated temperature, and then first-strand cDNA was synthesized by using random hexamer primers and M-MuLV Reverse Transcriptase (NEB). Following cDNA synthesis, the second-strand cDNA was synthesized using DNA polymerase I and RNaseH. Subsequently, the cDNA fragments were adenylated and ligated with adaptors. The ligation products were purified and then enriched using PCR amplification to create libraries. RNA-Seq libraries were sequenced using an Illumina HiSeq4000 platform at Novogene Bioinformatics Institute (Beijing, China).

The DEPC-treated lysis buffer, containing 150 mM of NaCl, 50 mM of Tris–HCl (pH = 7.4), 1% Triton X-100, 1 mM of ethylenediaminetetraacetic acid, 40 U/ml of ribonuclease inhibitor (TaKaRa, Kusatsu, Shiga, Japan, 2313Q), and proteinase and phosphatase cocktail inhibitors, was mixed well by agitation for 30 min. First, the rabbit anti-IGF2BP3 antibody was incubated with protein A magnetic beads pretreated with lysis buffer at a ratio of 1:100 for 2 h at 4°C with rotation. Then, the 1.5–2.0 × 10⁷ cell pellet was resuspended in 1 ml of lysis buffer after centrifugation at 1,000 rpm for 3 min. Subsequently, the samples were centrifuged at 12,000 rpm for 30 min, and the supernatant was removed to a new Eppendorf tube. For the RNA extraction of the INPUT sample, 10% of the supernatant (100 μl) was mixed with TRIzol reagent. The remaining supernatant (900 μl) was incubated with the magnetic beads–antibody complex overnight with rotation. After being washed six times with 1 ml of lysis buffer, the immunoprecipitants were directly mixed with TRIzol reagent in order to isolate the RNA as described above. All steps were performed at 2–8°C.
NEB Next Ultra Directional RNA Library Prep Kit was used as our preferred method in preparing the clusters of cDNA libraries. RNA sequencing was finally performed on Illumina Hi-Seq sequencing platforms.

Whole colon was dissected from G0- and G2-colonized ASF mice and homogenized by polytron in TRIZOL (Thermo Fisher) after removing feces. Total RNA was extracted by following the manufactural protocol of TRIZOL. 500 ng of total RNA was used for rRNA depletion (New England Biolabs, E6310), followed by directional library synthesis by using Next® Ultra™ Directional RNA Library Prep Kit (NEB E7420). Sequencing was performed by NextSeq500 (Illumina). A paired-end run was conducted to obtain 2 × 36-base reads. FASTQ files were imported to CLC Genomics Workbench (CLC-GW, v10.1.1, Qiagen), mapped to mouse reference genome (mm10), and quantified for 49,585 genes. For statistical analysis, pairwise comparison was conducted with Empirical Analysis of DGE tool in CLC-GW. Differentially expressed genes were determined by a false discovery rate-adjusted P-value of less than 0.05.