title: GSM3499767: WT 16DAF rep1; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total RNA was extracted from the pooled seeds of 30-40 siliques using the RNeasy plant mini kit (Qiagen) according to the manufacturer's instructions, followed by additional purification with sodium citrate to improve RNA quality. We obtained total RNA yields in the range of 1-3ug for ~50mg seeds. Using an Agilent Bioanalyzer, we confirmed that the RNA Integrity Number (RIN) for all samples was greater than 9. Total RNA (500 ng) was sequenced at the Genomics Core at the University of Kansas Medical Center. The mRNA fraction was enriched with oligo dT capture, sized, reverse transcribed into cDNA and ligated with the appropriate indexed adaptors using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). Following Agilent Bioanalyzer QC of the library preparation and library quantification using the Roche Lightcycler96 with KAPA SYBR Fast Universal qPCR kit (KAPA Biosystems), the RNA-Seq libraries were adjusted to a 4nM concentration and pooled for multiplexed sequencing. Libraries were denatured and based on qPCR results, diluted to the appropriate concentration, followed by clonal clustering onto the sequencing flow cell using the TruSeq Paired-End (PE) Cluster Kit v3-cBot-HS (Illumina). The clonal clustering procedure was automated using the Illumina cBOT Cluster Station. The clustered flow cell was sequenced on an Illumina HiSeq 2500 Sequencing System using the TruSeq SBS Kit v3-HS (Illumina) to obtain 100bp pair-end reads, which were trimmed and quality assessed before assembly against the Arabidopsis reference genome (Araport 11) using CLC genomics workbench (v 7.5.1).