Rna 1000 nano labchip kit

Mid-logarithmic phase algal cells were transferred into darkness overnight, followed by exposure to high light and nitrogen-depleted conditions with 5% CO₂. After 96 h, aliquots of cells were collected for transcript analysis. The total RNA of the algal cells was prepared using an RNA miniprep kit (CWBIO) and the quantity and purity were analyzed using the Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, USA), with an RNA integrity value of > 7.0. Poly(A) RNA was purified from 5 μg of total RNA using poly-T oligo-attached magnetic beads with two rounds of purification.
After purification, the mRNA was fragmented into small pieces with divalent cations at an elevated temperature. We then reverse-transcribed the cleaved RNA fragments to create the final complementary DNA (cDNA) library, following the protocol of the mRNA Seq Sample Preparation Kit (Illumina, USA). The average insert size for the paired-end libraries was 300 bp (± 50 bp). Finally, paired-end sequencing was performed on the Illumina Novaseq™ 6000 platform.

Total RNA was isolated from SW1353 and SW-1353-R cells for RNA sequencing. RNA quality and integrity were examined using a Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent). All samples with an RNA integrity number of less than 7 were excluded from the subsequent assay. After mRNA fragmentation and generation of a complementary DNA (cDNA) library, RNA sequencing was performed using an Illumina HiSeq 4000 and mapped using the HISAT package (http://ccb.jhu.edu/software/hisat2). EdgeR software was used to estimate the differentially regulated genes of all transcripts by calculating fragments per kilobase per million (FPKM). The differentially expressed genes were determined by log2 (fold change) >1 or log2 (fold change) <-1 values, and their statistical significance (p-value <0.05) was defined by R package software. The original data has been uploaded and provided with the GSE number (GSE239911).

Total RNA of the liver and terminal ileum was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) and purified using an RNA 1000 Nano LabChip kit (Agilent, Santa Clara, CA). The cDNA library was constructed using the mRNA-Seq sample preparation kit (Illumina, San Diego, CA). The cDNA library was then sequenced with an Illumina HiSeq 4000 (Illumina), and 150-bp paired-end reads were generated.
The raw sequence data were first processed using in-house Perl scripts to remove reads containing adapters, reads containing poly-N and low-quality reads. The clean reads were aligned to the UCSC (http://genome.ucsc.edu/) rat reference genome using HISAT2 (version 2.0.5), assembled using StringTie (version 1.3.3b), and merged to reconstruct a final transcriptome using Perl scripts (52 (link)). StringTie (version 1.3.3b) and featureCounts (version 1.5.0-p3) were then utilized to determine the expression levels of mRNAs by calculating fragments per kilobase of exon model per million mapped reads (FPKM) (53 (link), 54 (link)).
A differential transcription analysis of two groups was performed using the DESeq2 R package (version 1.20.0). The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with a P_adj of <0.05 identified by DESeq2 were assigned as differentially expressed transcripts.

Three samples from each N and R group were collected for transcriptome sequencing (three samples for each stage). Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA), and their quality was assessed using the Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with RIN (RNA integrity number) >7.0. Poly-T magnetic beads were used twice to purify poly(A) RNA from total RNA (5 μg). Following purification, mRNA was fragmented with divalent cations at elevated temperatures and then reverse transcribed to create the final cDNA library with an average insert size of 300 bp (±50 bp). Thereafter, paired-end sequencing was performed on the Illumina Novaseq™ 6000 platform (LC Sciences, TX, USA) following the manufacturer’s protocol.

We mixed all samples of different treatments (P + H, P + Fe, P + Mg, P + Cu, PVY + H, PVY + Fe, PVY + Mg, PVY + Cu) at 3 time points into one sample for full-length transcriptome analysis based on SMRT sequencing. Total RNA was extracted using a Trizol reagent (Invitrogen, CA, USA). The RNA quantity and purity were analyzed by Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0. Full-length cDNA was synthesized using a SMAR Ter™ PCR cDNA Synthesis Kit (Clontech, CA, USA). The generated cDNA was then re-amplified using PCR. After end repair, the SMRT adaptor with a hairpin loop structure was ligated to the cDNA. The cDNA library was then constructed via exonuclease digesting. After quality measurement of the cDNA library, SMRT sequencing was performed using the Pacific Bioscience Sequel platform (Biomarker Technologies Co. Ltd., Beijing, China). The reference genome of tobacco K326 (https://solgenomics.net/organism/Nicotiana_tabacum/genome) was used. The sequencing data were deposited in the SRA database at NCBI with the accession number PRJNA903693.

Total RNA was extracted using Trizol reagent (Invitrogen, CA, United States) as instructed by the manufacturer. The quantity and purity of total RNA were determined using the RNA 1000 Nano Lab Chip Kit (Agilent Technologies, Santa Clara, CA, United States) on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). Poly (A) RNA was purified from total RNA (5 μg) in two rounds using poly-T oligo-attached magnetic beads. Next, the mRNA was fragmented into small pieces at high temperatures using divalent cations, followed by reverse-transcription of the cleaved RNA fragments to create the final cDNA library following the protocol for the mRNA seq sample preparation kit (Illumina, San Diego, USA). The cDNA library was constructed by the Hangzhou Lianchuan Biotechnology Co., Ltd. (Hangzhou, China) and sequenced by Illumina Hiseq4000 with 150 bp paired-end reads. The root RNA sequencing analyses were performed with two biological replicates.

Three samples of HUCCT1 cells transfected with shHMGA1 or control vector were subjected to RNA-seq. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analyzed with a Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Poly(A) RNA was purified from total RNA (5 µg) using poly-T oligo-attached magnetic beads using two rounds of purification. Following purification, the mRNA was fragmented into small pieces using divalent cations at an elevated temperature. Then, the cleaved RNA fragments were reverse transcribed to create the final cDNA library in accordance with the protocol for the mRNA Seq sample preparation kit (Illumina, San Diego, USA), and the average insert size for the paired-end libraries was 300 bp (±50 bp). Then, we performed paired-end sequencing on an Illumina X10 at LC Sciences (USA) following the vendor’s recommended protocol. The RNA-seq data generated in this study have been deposited in the NCBI GEO database under the accession number GSE163759.