2100 bioanalyzer high sensitivity dna chip

Total RNA including miRNA was extracted from NHP serum samples using miRNeasy (QIAGEN). All extracted RNA was used in the library preparation following Illumina’s TruSeq -small-RNA-sample preparation protocols (Illumina, San Diego, CA, USA). Quality control analysis and quantification of the DNA library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Single-end sequencing 50 bp was performed on Illumina’s Hiseq 2500 sequencing system following the manufacture’s recommended protocols.

Total RNA was isolated and purified with a RNeasy Micro kit (QIAGEN, Hilden, Germany) and treated with DNAse (Promega Corporation, Madison, WI, USA). RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA integrity numbers of all samples were between 7.9 and 9.4. A poly (A) RNA-sequencing library was prepared by LC Sciences (Houston, TX, USA) following Illumina’s TruSeq-stranded-mRNA sample preparation protocol (Illumina, San Diego, CA). Poly (A) tail-containing mRNAs were purified using oligo-(dT) magnetic beads with two rounds of purification. After purification, poly (A) RNA was fragmented using divalent cation buffer in elevated temperature. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies). Paired-ended sequencing was performed on Illumina’s NovaSeq 6000 sequencing system (Illumina).

Library preparation and poly-adenylated RNA sequencing were performed by LC Sciences (https://www.lcsciences.com/). Briefly, RNA-seq paired end libraries were created using Illumina’s TruSeq-stranded-mRNA sample preparation protocol (Illumina, San Diego, CA). Integrity of RNA was checked using an Agilent Technologies 2100 Bioanalyzer. Two rounds of purification of poly(A) containing mRNAs were performed using oligo-dT magnetic beads. cDNA libraries were made and quality was assessed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing of the cDNA libraries was performed using llumina’s NovaSeq 6000 sequencing system. The sequencing resulted in paired 150 bp reads with approximately 6GB of data per run, resulting in a sequencing depth of approximately 40 million reads per sample.
Sequence data was submitted to NCBI sequence read archive (SRA) under the BioProject ID PRJNA767080.

We evaluated the lung transcriptome profiling of lung homogenates from 1-day-old in utero-exposed male and female mouse offspring (n = 4 per group) via RNA sequencing (poly A). Following the RNA extraction of the lung samples, the RNA was shipped to LC Sciences (Houston, TX, USA) to be further processed and sequenced. A poly (A) RNA sequencing library was prepared by LC Sciences following Illumina’s TruSeq-stranded-mRNA sample preparation protocol. The integrity of the RNA was verified with an Agilent Technologies 2100 Bioanalyzer. Quality control analysis and the quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina’s NovaSeq 6000 sequencing system. The sequencing depth was 150 bp paired-end, >40 million reads. Bioinformatics analyses were subsequently performed by LC Sciences. The differentially expressed mRNA were selected with a log2 |fold-change| > 1.5 and with a statistical significance of p < 0.05.

Total RNA was extracted using miRNeasy kit (Qiagen) following the manufacturer’s instructions. RNA-Seq libraries were constructed using the TruSeq sample Prep Kit V2 (Illumina). Briefly, 1 μg of purified RNA was poly-A selected and fragmented with fragmentation enzyme. After first and second strand synthesis from a template of poly-A selected/fragmented RNA, other procedures from end-repair to PCR amplification were done according to library construction steps. Libraries were purified and validated for appropriate size on a 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies.). The DNA library was quantified using Qubit and normalized to 4 nM before pooling. Libraries were pooled in an equimolar fashion and diluted to 10 pM. Library pools were clustered and run on Nextseq500 platform with paired-end reads of 75 bases, according to the manufacturer’s recommended protocol (Illumina). Raw reads passing the Illumina RTA quality filter were pre-processed using FASTQC for sequencing base quality control. Sequence reads were mapped to UCSC human genome build using TopHat and differential gene expression determined using Cufflinks 2.1.1 and Cuffdiff2.1.1 as implemented in BaseSpace. Sequencing data has been deposited to publically available GEO dataset GSE122953.