Truseq rna sample prep kit

cDNA libraries were prepared from high quality RNA (3 μg per sample) using an Illumina TruSeq RNA sample prep kit following the manufacturer’s instruction (Illumina, San Diego, CA, USA). Briefly, mRNA was isolated from total RNA and strands subsequently fragmented. First strand cDNA was synthesised using SuperScript^® II Reverse Transcriptase (Applied Biosystems Ltd., Life Technologies, Warrington, UK), second strand synthesis was subsequently performed using components supplied in the Illumina TruSeq RNA sample prep kit. Indexing adaptors were ligated to the cDNA which was then enriched through PCR. Final individual cDNA libraries were validated on the Agilent Bioanaylser 2100 using the DNA 1000 Nano Lab Chip kit, ensuring that library fragment size was ~260 bp and library concentration was >30 ng/μl. After quality control procedures, individual RNA-seq libraries were pooled based on their respective sample-specific-6bp adaptors and sequenced at 100 bp/sequence on an Illumina HiSeq 2000 generating single-end reads.

RNA-seq and de novo assembly were performed as previously described [11 (link)]. In brief, we extracted total RNA from the leaves of the collected Physostegia virginiana plant using an EASYspin Plus Complex Plant RNA Kit (Aidlab Biotech, Beijing, China) for subsequent RNA-seq. Ribosomal RNAs were removed using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). An RNA library was constructed using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol, followed by sequencing on the Illumina HiSeq X-ten platform (Biomarker Technologies, Beijing, China). Low-quality reads were filtered, and adapters of the paired-end raw reads were trimmed using the CLC Genomics Workbench 9.5 software (QIAGEN, Hilden, Germany). The clean reads were assembled de novo into contigs using the Trinity v2.3.2 program with default parameters (Broad Institute and the Hebrew University of Jerusalem, Cambridge, Massachusetts and Jerusalem, USA, and Israel), and K-mer was 25 bp [10 (link)].

Total RNA was extracted from each sample using an RNAprep pure Plant Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. The RNA concentration of each sample was measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The RNA quality was assessed using an Agilent2200 (Agilent Technologies, Santa Clara, CA, USA).
The sequencing library for each RNA sample was prepared using a TruseqTM RNA sample prep Kit (Illumina, San Diego, CA, USA), following the manufacturer’s protocol. Briefly, mRNA was purified using poly-T oligo-attached magnetic beads (Invitrogen,Carlsbad, CA, USA) from 5 μg total RNA. The mRNA was fragmented, and the RNA fragments were reverse transcribed and amplified to double-stranded cDNA. Index adapters were then ligated to the cDNA according to the protocol of the TruseqTM RNA sample prep Kit (Illumina). The library was quantified using a TBS-380 mini-fluorometer (Picogreen, Cohasset, MA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System, using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and a sequence length of 2*101 bp paired-end reads were generated.

Total RNAs of rapeseed embryo, maize embryo, and castor bean endosperm were extracted for transcriptome analyses. Plant tissues were grounded with liquid nitrogen and total RNAs were isolated following manufacturer’s instructions of the RNA extraction kit (9769, TaKaRa). RNA concentration was measured using nanodrop spectrophotometer (Nanodrop Technologies, Santa Clara, CA, USA) and integrity was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Subsequently, an Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) was used to build cDNA libraries.

RNA sequencing was performed according to previously published protocols with some modifications (25 (link)). Total RNA from freshly egressed TgTKL4-WT and TgTKL4-KO parasites was isolated using the RNeasy kit (Qiagen). The quality of total RNA samples was verified by using a BioAnalyzer (Agilent), followed by digestion with DNase I (NEB). rRNA was removed using the Ribo-Zero rRNA removal kit (human/mouse/rat; Illumina). Sequencing libraries were then generated using the TruSeq RNA sample prep kit (v2; Illumina) according to the manufacturer’s protocol. Libraries were amplified using the TruSeq cluster kit (v3; Illumina) and subjected to 50-bp single-end sequencing with the Illumina HiSeq 2000 system. Sequencing reads were aligned to the Toxoplasma GT1 reference genome (ToxoDB v.53; www.toxodb.org) using the STAR software package (v.2.7.1a, with default settings) (42 (link)). Filtered and normalized gene expression levels were calculated from the aligned reads using HTSeq v.0.13.5 (43 (link)). Differentially expressed genes were identified by linear modeling and Bayesian statistics using the limma package for R v.3.49.1 (44 (link)).

RNA of Gltscr1^−/− and Gltscr1^+/+ cardiac tissues collected from E12.5, E13.5, and E15 embryos were extracted, sequenced, and analyzed by bioacme (Wuhan, China). Each group collected three embryos from the same parent and were used for condition. The cDNA libraries were prepared from high-quality RNA using an Illumina TruSeq RNA sample prep kit following the manufacturer’s instructions (Illumina, San Diego, CA, USA). The individual RNA-seq libraries were pooled based on their respective sample-specific 6-bp adaptors and sequenced at 150-bp/sequence pair-read using an Illumina NovaSeq system. Clean Reads were mapping into the hg19 reference genome by STAR and quantified by RSEM. Differential expression genes were identified by DESeq2 in R. Benjamini–Hochberg false discovery rate method was applied to correct for multiple hypothesis testing. Genes with P < 0.05, fold change > 1.5, or fold change < 0.67 were defined as different expression genes as candidates for further analysis. Gene expression Heatmap was accomplished with R package pheatmap. Gene Ontology enrichment analysis was performed using DAVID. The results were visualized by the R package ggplot2 in R software. All raw and processed sequencing data generated in this study have been submitted to the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA820129.