Truseq mrna seq kit

Sequencing libraries from purified RNAs were prepared using the TruSeq mRNA-Seq kit with associated protocol (Illumina, Inc., San Diego, CA). Briefly, mRNAs were isolated via attachment to oligo(dT) beads, chemically fragmented to a mean size of 150 nt, and reverse transcribed into cDNA via random hexamer priming. Resulting double-stranded cDNA fragments were end-repaired to create blunt-end fragments, 3’ adenine-tailed, ligated with indexed TruSeq adapters (Illumina, Inc.), and PCR-amplified using TruSeq primers (Illumina, Inc.). Purified PCR-amplified libraries were checked for quality and quantity on a Bioanalyzer DNA 7500 chip (Agilent Technologies, Inc.) with 2100 Expert software before normalization and sample pooling. Sample pools of 10 nM were clustered and sequenced on the HiSeq 2000 (WCR) or 2500 (WCR, FAW, SGSB) system with TruSeq Sequencing By Synthesis Rapid v3 (WCR) or v4 (WCR, FAW, SGSB) chemistry (Illumina, Inc.), as per manufacturer’s instructions. Samples were sequenced single-read, fifty cycles per read, to a minimum depth of five million reads per sample and a target depth of ten million reads per sample.

After the plants reached V3, six leaf punches were taken from the 3rd leaf of each plant. These were immediately frozen in liquid nitrogen and stored at −80 °C until processed for RNAseq analysis. Total RNA was isolated from frozen tissues using a Qiagen RNeasy Kit for total RNA extraction. Libraries from total RNA were then prepared using the TruSeq mRNASeq Kit (Illumina) and sequenced on an Illumina HiSeq 2500 system.

Synchronously developing Xenopus tropicalis embryos were obtained by in vitro fertilization using standard methods. Stage 10–10.25 gastrula embryos were manually dissected into five fragments representing ectoderm, dorsal mesoderm, lateral mesoderm, ventral mesoderm and endoderm (Blitz et al., 2016 ) and RNAs were isolated after homogenization. The RNA samples were subjected to polyA+ selection and library production according to the Illumina Tru-Seq mRNA-seq kit. Libraries were ligated using bar-coded adaptors and subjected to 50-bp single end sequencing on an Illumina HiSeq2000 instrument (Blitz et al., 2016 ). Dissection RNA-seq datasets can be found in Blitz et al. (2016) .

RNA extraction was conducted using the Clontech NucleoSpin RNA XS kit (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s protocol. For sequencing library preparation, 1.5 μg of total RNA was utilized with the TruSeq mRNA Seq kit (Illumina, San Diego, CA, USA). The cDNA reads were then sequenced on a HiSeq 2500 platform (Illumina, San Diego, CA, USA) using a 101 bp paired-end 30-million read protocol. The short reads in fastq format underwent quality control using FastQC (Andrews S. (2010). FastQC: a quality control tool for high-throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 26 April 2010). Subsequently, reads were quantified at the transcript level using Kallisto [49 (link)] pseudoalignment against Ensembl human reference GRCh38 and gene annotation file and aggregated to gene-level counts using R package “tximport” version 1.32.0 [50 (link)]. The raw RNAseq count matrix data were further normalized and processed using the “limma” package version 3.50.0 [51 (link)] from Bioconductor to generate differential expression profiles. Inflamed samples were compared with non-inflamed samples, and genes with Benjamini–Hochberg-adjusted p values below 0.05 were considered significantly differentially expressed.

Total RNA was isolated with Qiagen RNA Isolation Kit (Qiagen). The DNaseI Enzyme Kit (Roche) was used to remove DNA from the RNA samples. Complementary DNA was synthesized from the total RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). PCR amplifications were performed using the TaqMan probe‐based detection system according to the manufacturer's instructions (Applied Biosystems). Primers and probes are shown in Table S2. Relative quantification values were determined using the difference in Ct from the target genes and the reference gene, maize UBIQUITIN5.
RNA sequencing (RNA‐seq) was performed as described (Shi et al., 2015). In brief, total RNA was isolated from frozen maize tissues and used to prepare sequencing libraries using the TruSeq mRNA‐Seq Kit (Illumina), and sequenced on the Illumina HiSeq 2000 system with Illumina TruSeq SBS v3 reagents. On average, 10 million sequences were generated for each sample. The resulting sequences were trimmed based on quality scores and mapped to the maize B73 reference genome sequence V2 and normalized to reads per kilobase of transcript per ten million mapped reads. The generated data matrix was visualized and analysed in GeneData Analyst software (Genedata AG, Basel, Switzerland).

RNA was collected from MDA-MB-436-dCas9-KRAB cells subjected to CRISPRi targeting as described above. RNA-seq library preparation and sequencing was performed by the Novogene Company (novogene.com). Libraries were prepared using the Illumina TruSeq mRNA-seq kit following the manufacturer’s instructions and were sequenced by paired-end 150 bp sequencing on the Illumina Novaseq 6000 platform. RNA-sequencing was aligned to the hg38 genome using STAR 2.7.6a and quantified with Salmon 1.4.0. All samples were evaluated to ensure sequencing passed acceptable quality thresholds for analysis by utilizing the Picard 2.22.4 CollectRnaSeqMetrics function and FastQC. Differential expression analysis was performed using the DESeq2 package in R. Differentially expressed genes were defined as any gene with an adjusted p-value < 0.1 and a mean expression >20 (DESeq2 mean of normalized counts for all samples). RNA-seq data were uploaded to GEO under the following accession number GSE185318.