Vahts universal v6 rna seq library prep kit

The total RNA of Huh-7 and SOX9^KO Huh-7 cells was extracted using TRIzol Reagent (Life Technologies, 15596026). The RNA quality (RNA integrity number, RIN) was checked with an Agilent Bioanalyzer, and confirmed a high integrity RIN > 7 for all samples. RNA sequencing libraries were prepared with the VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme NR604-02) according to the provided instructions. Sequencing was performed in the single read mode using an Illumina Novaseq 6000. Sequenced reads were aligned to the GRCh38 reference genome with TopHat2 (v 2.0.14). RNA sequencing data can be accessed publicly on GEO under the accession number GSE214335.

Total RNA was extracted using the TRIzol reagent (Invitrogen, USA, 15596–026) according to the manufacturer's protocol. Then the libraries were constructed using VAHTS Universal V6 RNA‐seq Library Prep Kit (Vazyme, China, NR604) according to the manufacturer's instructions. Pooled libraries were sequenced on a HiSeq 4000 sequencer (Illumina, San Diego, CA, USA) generating 30 million paired‐end (100 bp) reads per sample. Gene quantification was performed using the script htseq count in the Python software package HTSeq (v0.9.1). Genomic characteristics were defined using GENCODE v28, where each gene is considered a combination of all its exons. Gene counts were normalized for sequencing depth, and genes with a mean expression less than 10 were removed. Missing values were filled in with the k‐nearest neighbors' method using the “impute. knn” function in the R software package “impute” (v1.52.0). Two data sets were generated, one containing gene counts normalized for sequencing depth in each sample (raw counts), and the other was further processed with variance‐stable transformation (VST) using the R software package “DESeq2”.

RNA sequencing was performed by OEbiotech (Shanghai, China). Briefly, total RNA was extracted from C666-1 cells, C666-1 cells treated with P8 (C666-1-P8) and C666-1 cells infected with p53-R280T lentivirus and treated with P8 (C666-1-R280T-P8) using TRIzol reagent. A VAHTS universal V6 RNA-seq library prep kit (Vazyme) was used to construct the RNA library, followed by sequencing on an Illumina NovaSeqTM 6000 platform (Illumina, CA, USA). The differentially expressed genes (DEGs) were identified based on |Fold Change | >1.5 and p value < 0.05, and differential expression analysis was performed using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the significantly enriched terms through R (v3.2.0). Gene set enrichment analysis (GSEA) was carried out using GSEA software.

Total RNA was extracted using the Ambion-1561 mirVana ^TM miRNA Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. RNA purity and quantification were measured using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). RNA integrity was determined via 1.0% agarose gel electrophoresis, and only samples meeting the following criteria were used for library construction: RIN (representing RNA Integrity Number) value ≥ 7, 28S/18S ≥ 0.7, OD260/280 value between 1.8~2.2, and a concentration of ≥50 ng/μL. The Ribo-off rRNA Depletion Kit (Vazyme, Nanjing, China) was employed to eliminate ribosomal RNA, and then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

The TRIzol (Thermo Scientific, Waltham, MA, USA) reagent was used to extract total RNA from the epididymal cauda. RNA purity, quantification, and integrity were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. The libraries were sequenced on an llumina Novaseq 6000 platform. Raw reads were processed using fastp [21 (link)] to de-link, remove low-quality reads, and exclude low-quality bases from 3′–5′ caudas, Q30, and GC content in order to conduct decaudaed evaluation obtain clean reads. The clean reads were mapped to the reference genome (LU_Bosgru_v3.0) using HISAT2 (Johns Hopkins Hospital, Baltimore, USA) [22 (link)]. FPKM3 [23 (link)] of each gene was calculated, and read counts of each gene were obtained using HTSeq-count4 (http://www-huber.embl.de/HTSeq, accessed on 28 October 2022) [24 (link)]. Principal component analysis (PCA) was performed using R (v3.2.0, Shenzhen, China) to evaluate biological duplication of the sample.

The retinas of C57BL/6J mice were collected directly into TRIzol (Thermo Fisher). Total RNA was extracted according to the manufacturer’s instructions, and RNAs from 14 retinas were pooled together for each MeRIP-seq. MeRIP-seq was performed according to a published protocol (Dominissini et al., 2013 (link)). Briefly, PolyA-mRNA was purified using a Dynabeads mRNA DIRECT Purification kit (Thermo Fisher). mRNA was fragmented in fragmentation buffer heated at 94℃ for 5 min. Fragmented mRNA was immunoprecipitated against an m⁶A antibody (Synaptic Systems) bound to Protein G Dynabeads (Thermo Fisher) at 4°C for 2 hr, washed, and eluted by competition with free m⁶A (Sigma). After recovery of precipitated m⁶A-mRNA, sequencing libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme). The libraries were sequenced on an Illumina HiSeq platform on a 150 bp paired-end run. Raw data were filtered to remove low-quality reads to obtain clean reads. Clean reads were mapped to the mouse genome (GRCm38, mm10) using BWA mem v0.7.12. m⁶A peaks were determined using MACS2 v2.1.0 with the q-value threshold of enrichment set at 0.05. Motif analysis was done using MEME. GO term analysis was performed using clusterProfiler package (Yu et al., 2012 (link)).

Total RNA was isolated and purified using TRIzol (Life Services Inc., Glendale, CA, USA) following the manufacturer’s procedure. After the quality inspection of Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoPhotometer^® (IMPLEN GmbH, Munich, Germany), poly (A) RNA was purified from 1μg total RNA using VAHTS^® mRNA Capture Beads with Oligo (dT) (Vazyme, Nanjing, China) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using VAHTS^® Universal V6 RNA-seq Library Prep Kit (Vazyme, Nanjing, China) at 94°C for 8 min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA with reverse transcription reagent, which was next used to synthesize U-labeled second-stranded DNAs. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, size selection was performed with VAHTS^® DNA Clean Beads (Vazyme, Nanjing, China). Then the ligated products were e amplified with PCR. Finally, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (Illumina, Inc., San Diego, CA, USA) following the manufacturer’s recommended protocol.