Truseq total rna sample prep kit

RNAs from H157 cells stably expressing YAP, YAP/TEAD4-FL, YAP/TEAD4-S, YAP/RBM4 and control vectors were purified using Trizol method, and subsequently cleaned using the RNAeasy Kit (Qiagen). The RNAs were digested in column with RNAse-free DNAse as per the manufacturer's instruction. Total RNA not exceeding 3 μg was further used to purify polyadenylated RNA using the Illumina TruSeq Total RNA Sample Prep kits. We used the Ribo-Zero Human to remove the cytoplasmic rRNA. The mRNA purified was further analysed using the Bioanalyzer (Agilent Technologies) before generation of cDNA library with bar-coded ends. RNA-seq libraries were robotically prepared using the Illumina TruSeq Total RNA Sample Prep kits according to the manufacturer's protocol. The RNA-seq data set was deposited to the Gene Expression Omnibus with accession number GSE80372.
To estimate the gene expression levels, we used RSEM package and bowtie2 (refs 51 (link), 52 (link)) to align all reads to human reference genome (UCSC hg19 version). Then, we provided a fragment length distribution with options of ‘–fragment-length-mean 75' and ‘–fragment-length-sd 10' to calculate transcript expression levels. Subsequently, we used EBSeq tool53 (link) to examine differential expression genes of pair-wise comparison based on empirical Bayesian methods.

The DRGs from the Sham, SNI, and SNI with mitochondrial injection therapy (SNI + Mito) groups were subjected to RNA sequencing. Each group contained 2 samples, with each sample consisting of 6 DRGs (L3/4/5 from 2 mice). The Qiagen RNeasy Plus Universal Kit was utilized to extract total RNA from DRGs, following to the manufacturer’s recommended procedure. The Illumina TruSeq Total RNA Sample Prep Kits were utilized to prepare the libraries. The quantification of barcoded libraries was conducted before sequencing on the Illumina Hi-Seq 2000 system. The raw FASTQ data files of paired-end reads were then gathered for further analysis. To align with the UCSC mouse reference genome assembly mm9, TopHat was employed on the generated FASTQ files. The TopHat software utilized the high-throughput short read aligner, Bowtie, for mapping the reads to the reference genome. The aligned reads underwent processing using Cufflinks 2.0.0. Transcript abundance was quantified using fragments per kilobase of exon per million mapped fragments (FPKM). Differential expression analysis comparing different groups was conducted with the Cuffdiff module, and genes exhibiting a p-value < 0.05 were identified as displaying differential expression.

Total RNA was extracted from hippocampal tissues using TRIzol reagent (Invitrogen), and 3 μg of total RNA was treated with DNase I (Worthington Biochemical), followed by RNA purification using RNA Clean and Concentrator-5 Kit (Zymo Research) according to the manufacturer’s protocols. Purified RNA was subjected to quality control (Fragment Analyzer). cDNA libraries were prepared using Illumina TruSeq Total RNA Sample Prep Kits (Illumina) and High-Throughput 3′Digital Gene Expression method for 3 and 13 months mouse brain, respectively. Libraries were sequenced on the Illumina HiSeq 2000 platform at the MIT BioMicro Center. The collapsed raw fastq reads were aligned by Tophat2, and further processed by Cufflinks 2.0.0 with UCSC mm9 reference gene annotation to determine transcript abundances. The mean yield per sample was 14,217,977 sequencing reads. A gene was considered differentially expressed with a fold change ≥1.5 and a statistical significance of P < 0.05. GO analysis of DEGs was performed using gene set enrichment analysis^{31 ,32} for GO biological process (MSigDB, Broad Institute). TF-binding motif analysis of DEGs was conducted using the HOMER software⁴⁰ . Sequencing datasets are available to the public in the GEO Data Bank under accession numbers GSE115437 and GSE147407. The lists of DEGs can be found in Source Data file.