Agilent bioanalyzer rna nanochip

Total RNA was isolated using a NucleoSpin RNA Plant (Macherey-Nagel GmbH & Co., KG, 52355 Düren, Germany) minikit for RNA extraction and treated with RNase-free DNase. Three biological replicates for each of the time points and conditions were used. RNA quality (RNA integrity number—RIN > 8.0) was assessed using an Agilent Bioanalyzer RNA nanochip (Agilent, Wilmington, DE, USA). Sequence libraries were prepared using a TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. Both quality and insert size distribution were assessed using an Agilent Bioanalyzer DNA 1000 chip. Sequence libraries were quantified using qPCR (P5/P7 primers), pooled in equimolar concentration, and analyzed on an Illumina NextSeq500 generating 2 × 150 nt paired-end (PE) reads. The generated sequences were deposited in the NCBI (National Center for Biotechnology Information) PRJNA797248.

RNA stored in Trizol was extracted using the standard Trizol protocol followed by a further clean up step using membrane (RNeasy Micro Kit, Qiagen, USA). RNA was quantified using a NanoDrop spectrometer (NanoDrop Technologies), and quality of the RNA was determined by using Agilent Bioanalyzer (RNA Nano Chip, Agilent), and Qubit 2.0/3.0 fluorometer. RNA samples isolated from MDM were sequenced on an Illumina HiSeq 2000 platform generating readings of 75 bp in length in paired-end, for each sample three experimental replicates. Sequence analyses were performed at the University of Iowa DNA core facility.

The RNeasy Fibrous Tissue Mini kit (Qiagen) was utilized for total RNA extraction, using ceramic beads (MP Biomedicals) and TissueLyser-II for homogenization of tissue. Preparation of total RNA, washes and elution were performed according to kit protocol. The Agilent Bioanalyzer RNA-nano chip (Agilent) was used for evaluation of RNA integrity, and Qubit RNA kit (Life Technologies) for RNA quantification. Extracted RNA was stored at − 80 °c until library preparation, evaluation and sequencing, which were performed as previously described^{10 (link)}.

Gene expression profiling was performed by Affymetrix GeneChip® Human Gene 1.0 ST arrays (at one time point in the HMEC growth phase and two time points in the vHMEC phase referred to as early and late vHMEC, respectively), Taqman Low-Density miRNA arrays and RNA-seq.
RNA was extracted using TRIzol® reagent (Life Technologies, Carlsbad CA, USA) according to manufacturer’s protocol and quality assessed on the Agilent Bioanalyzer RNA Nano chip (Agilent Technologies, Santa Clara CA, USA) to ensure an RNA integrity number (RIN) of >9. RNA labelling and hybridisation to Affymetrix GeneChip® 1.0ST expression arrays was performed at the Ramaciotti Centre for Gene Function Analysis (UNSW, Sydney, Australia). Data analysis was performed using aroma.affymetrix [63 ]. Robust Multi-Array (RMA) normalisation to summarise gene-probe intensities and differentially expressed genes were identified using limma [64 ].

RNA from each collected sample was extracted using a NucleoSpin RNA Plant (Macherey-Nagel GmbH & Co. KG, 52355 Düren, Germany) and treated with RNase-free DNase. RNA quality (RNA Integrity Number (RIN) > 8.0) was evaluated using an Agilent Bioanalyzer RNA nanochip (Agilent, Wilmington, DE). RNA-Seq libraries were independently prepared for three pools representing the three different populations. Each pool was composed by equal leaf amount of three specimens. Sequencing library was prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) according to manufacturer’s specifications; quality and insert size distribution were evaluated using Agilent Bioanalyzer DNA 1000 chip. Sequencing libraries were quantified using qPCR and sequenced in the same lane on an Illumina HiSeq. 1000 generating 2 × 100 nt paired-end reads.

Total RNA was extracted using TRIzol reagent (Invitrogen) from three biologically-independent samples of (i) in vitro-cultivated leptospires or (ii) leptospires cultivated in DMCs (2 rats per sample) for 10 days as described above. Purified RNA was treated with Turbo DNAfree (Ambion, Inc. Austin, TX) as previously described [108] (link) to remove contaminating genomic DNA. The integrity of DNase-treated RNAs use for RNA-Seq were assessed using the Agilent Bioanalyzer RNA NanoChip (Agilent Technologies, Wilmington, DE) to ensure that each had an RNA integrity (RIN) value ≥8. One-hundred ng of total RNA was used for library generation according to Illumina standard protocols (TruSeq RNA Sample Preparation Guide, Low-Throughput Protocol, Part # 15008136 Rev. A). cDNAs were normalized using a duplex-specific nuclease (DSN) approach according to the DSN Normalization Sample Preparation Guide, Early Access Protocol, Part # 15014673 Rev. C, which decreases the prevalence of highly abundant transcripts, such as rRNAs. 76-bp paired-end sequencing was carried out by Sequensys (Prognosys Biosciences, La Jolla, USA) on an Illumina Genome Analyzer IIx according to the manufacturer's instructions.