Truseq rna sample prep kit v2 set a

Total RNA was extracted from SLE-iPSCs and Control-iPSCs using Trizol (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. The concentration and quality of total RNA were measured by the UV absorbance at 260 nm and 280 nm (A260/280) and checked by gel electrophoresis. The polyA mRNAs were selected by RNA Purification Beads (Illumina, SanDiego, CA). MicroRNA isolation was carried out from the total RNA using mirVana™ microRNA isolation kit (Ambion, Austin, TX) according to the manufacturer's instructions. Total mRNA and miRNA isolated from three independent cultures were, respectively, pooled for subsequent library construction and sequence analysis. The libraries of mRNA and miRNA were constructed by using the Illumina TruSeq RNA Sample Prep Kit v2-Set A and TruSeq Small RNA Sample Prep Kit Set A, respectively, sequenced by the Illumina HiSeq™ 2000 System. Adaptor sequences were subsequently trimmed to clean full-length reads and formatted into a non-redundant FASTQ format. The high-quality clean reads were mapped to the reference human genome using the SOAP (version 2.0) software. Sequences that perfectly matched the genome along their entire length were considered for next analyses. Differential expression of mRNA and miRNA between SLE-iPSCs and Control-iPSCs were calculated by relative expression analysis.

On dry ice (−80 °C), replicate vials were pooled and male flies sorted into chilled sterile microcentrifuge tubes containing 110 μl of 1.4 mm lysing matrix (MP Biomedicals), 500 μl buffer RLT (Qiagen), and 5 μl β-mercaptoethanol. Flies were immediately homogenized at 4.0 m/s for 30 s on a Fastprep-24 (MP Biomedicals) and frozen at −80 °C. RNA was isolated using the RNeasy mini kit (Qiagen) and prepared for sequencing with the TruSeq RNA Sample Prep Kit v2 – Set A (Illumina RS-122-2001, Additional file 11: Table S7). The samples were sequenced (50 bp single end reads) on the Illumina HiSeq 2500 platform in multiplexed pools of 6 samples per lane.

RNA-seq was used to identify common DEG transcripts among root tissues of four N-stress tolerant genotypes (San Chi San, China17, KS78, and the high-NUE bulk) and three sensitive genotypes [CK60, BTx623 (reference genome), and low-NUE bulk] grown under N-stress. The experimental process is summarized as follows: RNA libraries were prepared from 4 μg total RNA using the Illumina TruSeq RNA Sample Prep Kit v2 - Set A (RS-122-2002) according to the manufacturer’s instructions. Libraries were analyzed and measured by gel electrophoresis and NanoDrop 1000 Spectrophotometer to a concentration of 10 nM each. Four indexed libraries were pooled into one lane and clusters generated at 8 pM concentration were sequenced on the Illumina Genome Analyzer IIx (GAIIx; Illumina, Inc., San Diego, CA) using three 36-cycle sequencing kits to read 76 nucleotides of sequence from a single end of each insert, by standard multiplexing v8.3 protocol.