Ovation rna seq system v2 kit

The crypts, villus and MSCs were isolated from mouse intestine respectively. Then RNA of these cells was extracted by RNeasy mini kit (74,104, Qiagen) as manuals. cDNA libraries were conducted with the Ovation RNA-Seq System V2 kit (NuGEN) and subjected to high-throughput sequencing on an Illumina Novaseq PE150 platform. RNA-seq was carried out with two biological replicates. Sequencing reads were aligned to mouse genome reference (mm10) using STAR (Spliced Transcripts Alignment to a Reference) with default parameter. Differential expression analysis was performed using the R package DESeq2 based on gene counts data. Differentially expressed genes were identified with Log₂FoldChange absolute value > 1 and p value < 0.01.

RNA from freshly isolated intestinal crypts was converted into cDNA libraries using the Ovation® RNA-Seq System V2 kit (NuGEN). High-throughput sequencing was performed using Illumina HiSeq X10 for 3 biological replicates, respectively. For each sample, the RNA-seq data was mapped to mm10 genome by TopHat v1.4.1^{58 (link)} with no more than 2 mismatches, and then only the uniquely mapped reads were used to estimate the expression values in gene level by RPKM^{59 (link)}. Statistical significant test of differentially expressed genes was performed by DEseq with R. Genes with absolute log2-transformed fold changes greater than 1.7 were regarded as differentially expressed genes and a threshold of p value < 0.01 was used. Hierarchical clustering of log2-transformed RPKMs was generated by Cluster 3.0 and visualized by Java TreeView. The raw NGS data were deposited to the NCBI SRA database under accession number (SRP148616).

RNAseq libraries were prepared with Ovation RNAseq system v2 kit (NuGEN). In this method, the total RNA (50 ng or less) is reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating and the second-strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′-end of the primer by displacement of the existing forward strand. Random hexamers are then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA amplified cDNA were made using the Ultralow v2 library kit (NuGEN). The RNAseq libraries were analyzed by Bioanalyzer and quantified by qPCR (KAPA). High-throughput sequencing was done using a HiSeq 2500 instrument (Illumina). Libraries were paired-end sequenced with 100 base reads.
RNAseq data from this study are available in GEO under accession number GSE186036.

RNA was amplified using the Ovation RNA-Seq System V2 kit (NuGEN Technologies, San Carlos, CA, USA, lot no. 1307479-B) according to manufacturer’s protocol, yielding an average of 7.3 ± 0.9 µg double stranded cDNA per sample using 20 ng RNA input. Even distribution of sequencing reads along the length of each transcript was ensured through combined use of oligo(dT) hexamer and random primers.

The RNA sequencing was performed by the Genome Technology Center at the NYU Langone Medical Center. 10 ng of total RNA was used for library prep, and cDNA was amplified by using Nugen Ovation RNA-Seq System V2 kit (Part No. 7102-32), 100 ng of Covaris-fragmented cDNA were used as input to prepare the libraries, using the Ovation Ultralow Library system (Nugen, Part 0330-32), and amplified by 10 cycles of PCR. The samples were mixed into two pools and run in two 50-nucleotide paired-end read rapid run flow cell lanes on the Illumina HiSeq 2500 sequencer.