Truseq rna sample preparation kit v2

Total RNA were isolated from samples using Trizol (Ambion, Austin, TX, USA) as per the manufacturer’s protocol. RNA concentration, purity and integrity were first measured with NanoDrop ND-1000 UV–vis Spectrophotometer (NanoDrop Technologies) followed by RNA Nano 6000 Assay kit on Bioanalyzer 2100 system (Agilent Technologies). cDNA libraries were generated from total RNA with Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA, USA) following manufacturer's instructions. Quality of sequencing libraries was assessed with Bioanalyzer 2100 system. Sequencing was performed for total 12 sequencing libraries using Illumina HiSeq 2500 and 100 bp paired sequencing reads were produced.

A single head per each inoculated and mock-inoculated plant was collected at 48 h post inoculation and flash frozen in liquid nitrogen. The head tissues were ground to fine powder in an RNAse-free mortar precooled with liquid nitrogen. The RNA from the rachis was processed separately from the palea and lemma and they were pooled in 1:1 ratio for RNA-sequencing. RNA was extracted using Qiagen RNeasy Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The purity of RNA was tested using a NanoDrop ND8000 (Thermo Scientific, Wilmington, USA) and samples with an A260/280 ratio less than 2.0 were discarded. The quantity of RNA was determined using a Qubit® 2.0 Fluorometer (Grand Island, NY, USA) and a Qubit™ RNA broad range assay kit (Invitrogen, Carlsbad, USA) following the manufacturer’s protocol. The integrity of RNA was determined using an Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano Kit (Agilent Technologies Inc., Santa Clara, USA).
Total RNA (~ 1 μg) for each sample was used for library preparation using Illumina TruSeq® RNA sample preparation v. 2 kit (Illumina, San Diego, USA). The samples were sequenced (2 × 125 cycles, paired-end reads) on the HiSeq 2500 (Illumina, San Diego, USA) using the TruSeq SBS v3-HS 200 cycles Kit (Illumina, San Diego, USA).

To extract and quantify RNA, total RNA was extracted from cultured microglia using TRIzol Reagent (Life Technologies). To obtain enough RNA, same treatment cells were pooled in one replicate. RNA quantity and quality (RNA integrity number, RIN) was determined by using a RNA Nano Chips (Agilent RNA 6000 Nano Chips) with Agilent 2100 BioAnalyzer. All samples had a RIN-value ranging from 6 to 8.5, except for one sample having RIN = 5.5 but an acceptable 84% of transcripts mapped, which did not affect the read count for this sample.
A total of eight samples from four set of replicates were selected for RNA sequencing at high throughput, of which three replicates were derived from in vivo control fetal sheep and one replicate was from in vivo LPS-exposed (second hit) fetal sheep. RNAseq libraries were prepared by using Illumina TruSeq RNA Sample Preparation v2 kit (Illumina) and quality control was performed on the BioAnalyzer. Single-end 50-bp sequencing was performed at high throughput on an Illumina HiSeq2500 at the CHU Ste-Justine Core Facility Sequencing Platform. Raw data and RNAseq data discussed in this publication were deposited on NCBI and are accessible online with the GEO accession number GSE71037.

For the purpose of the RNA-seq analysis, RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) from a bulk of 100 shoot tips per replicate (three) per sampling point from both WT and wrky22.1 mutant shoot tips and treated with DNase. For mRNA purification and poly(A) selection, the Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA, USA) was used. Library preparation was performed using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI, USA) following the manufacturer’s protocol. Quality assessment of the libraries was done using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Cluster generation of the prepared libraries was performed using the cBot (Illumina) and TruSeq SR Cluster Kit v3-cBot-HS (Illumina) following the manufacturer’s instructions. The concentration of libraries loaded in the flowcells was 12 pM, followed by sequencing on a HiSeq 2500 instrument with the TruSeq SBS Kit v3-HS (Illumina) for 50 cycles. Image analysis and base calling were performed using the Illumina pipeline v 1.8.

We used single-cell and bulk RNA-seq data generated by Kim et al. [28 (link)], which have been deposited in the Sequence Read Archive (SRA, NCBI) using the Gene Expression Omnibus (GEO) (GSE69405). The RNA-seq data for the primary matched normal (pN) and mouse xenograft (xenoT) samples can be accessed in the GEO by GSE70968. According to Kim et al. [28 (link)], single-cell capture and cDNA amplification were performed using a SMARTer kit (Clontech, Mountain View, CA) on a 17–25 μm microfluidic chip (Fluidigm, San Francisco, CA). Sequencing libraries were generated using a Nextera XT DNA Sample Prep Kit (Illumina, San Diego, CA) for single cells or a TruSeq RNA Sample Preparation v2 kit (Illumina) for bulk RNA samples, and sequencing reads were generated using HiSeq2500 sequencing system in the 100-bp paired-end mode using a TruSeq Rapid PE Cluster kit and a TruSeq Rapid SBS kit (Illumina). RNA-seq reads were filtered at Q33 using Trimmomatic-0.30 [30 (link)] and were aligned to a human genome reference (hg19) using Tophat2 [31 (link)]. Transcripts were estimated as fragments per kilobase of transcript per million mapped reads (FPKM) using Cufflinks [31 (link)].

Complementary DNA (cDNA) libraries were generated from the RNA of BEAS-2B cells expressing empty vector or NUPR1 overexpression vector using a TruSeq RNA Sample Preparation V2 Kit (Illumina). Library preparations were validated with the Agilent Bioanalyzer using the DNA1000 kit. We adjusted the library concentrations to 4 nM. The libraries were then pooled for multiplex sequencing. Pooled libraries were denatured and then diluted to 15 pM. The diluted libraries were clonally clustered onto the sequencing flow cell using the Illumina cBOT Cluster Generation Station and a TruSeq Paired-End Cluster Kit v3-cBot-HS. We performed the sequencing using an Illumina HiSeq2500 Sequencing System using a TruSeq SBS Kit v3-HS. Raw sequencing reads were mapped to the human genome reference (GRCh37.71/hg19) using Bowtie aligner (0.12.9) with v2 and m1 parameters. Mapped reads were subsequently subjected to PCR duplicate removal prior to gene model assignment using featureCounts package. To identify significant differentially expressed genes, the raw reads of each sample from experimental and control groups were normalized and compared based on experimental design using default methods with the edgeR package (3.4.2). The FDR adjustment was employed for multiple hypothesis tests, and an appropriate FDR cutoff was applied to select significant differentially expressed genes for analysis.