Next ultra directional rna library prep kit for

RNA was extracted from 10‐week‐old VHL^−KO; Setd2^−KO and VHL^−KO mice PTECs. For the construction of sequencing libraries, New England Biolabs’ Next Ultra Directional RNA Library Prep Kit for Illumina (Ipswich) was utilised. Afterward, Illumina sequencing was performed with paired‐ends 2 × 150 as the sequencing mode. Differential gene expression was analysed using the DESeq2 package. The statistically significant DE genes were obtained by a p‐value threshold <.05 and fold change ≥1.5. All differentially expressed mRNAs were selected for Gene Ontology (GO) analysis.

Overnight cultures were inoculated in 100 ml of fresh LB medium to bring the initial optical density (OD) of the culture to 0.03, and the flasks were incubated at 37°C with shaking at 200 rpm. Samples were collected at the maximum growth rate, and two biological replicates were performed for each sample. The samples were immediately processed for total RNA isolation using the TRIzol method (15596018; Invitrogen). DNase-treated RNA was depleted of rRNA using the Ambion MICROBExpress kit (AM1905). Libraries were prepared for RNA sequencing using the New England Biolabs (NEB) NextUltra directional RNA library prep kit for Illumina (New England Biolabs), according to the manufacturer’s protocol, and single-end sequencing for 50 cycles was done using the Illumina HiSeq 2500 platform.

For each treatment, six larvae were randomly selected for RNA extraction, and one larva represented one replication for RNAseq analyses. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and concentration were checked on 1% agarose gels and Agilent 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA). The cDNA library was constructed following the manufacturer’s instructions for the NEB Next Ultra Directional RNA LibraryPrep Kit for Illumina (NEB, Ipswich, MA, USA). In brief, the mRNA was enriched by Oligo (dT) magnetic beads and then fragmented using a fragmentation buffer. These fragments were used to synthesize the first-strand cDNA and the second cDNA. End-repair was performed on the synthesized cDNA, followed by dA-tail and adaptor ligation. Standard AMPure XP (Beckman Coulter, Inc., Brea, CA, USA) was used to select DNA fragments of preferentially 150–200 bp in length. The constructed cDNA libraries were sequenced using an Illumina HiSeq platform with the paired-end reads in the Core PE150.

Total RNA was isolated using TRIzol reagent (Ambion, USA) following the manufacturer’s protocol. After evaluation of purity, concentration, and integrity, 3 μg of RNA per sample was used in library preparation for RNA sequencing. The rRNA was removed using the Ribo-zero rRNA Removal Kit (Epicentre, USA), and the rRNA-depleted sample was subsequently used to generate sequencing libraries using the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s protocols. In brief, the rRNA-depleted RNA samples were subjected to RNA fragmentation, first strand cDNA synthesis with random hexamer primers, second strand cDNA synthesis with dUTP, adenylation of 3’ ends, barcoded adapter ligation, purification, and amplification using PCR to generate sequencing libraries. After cluster generation, libraries were sequenced on an Illumina HiSeq 4000 platform by Novogene (Beijing, China).