title: GSM1147579: OMA3.01 RNA-seq replicate 2; Avena sativa/Zea mays addition line; RNA-Seq
library_construction_protocol: Leaves were collected and ground into fine powder in liquid nitrogen.  The resulting powder was suspended in nuclear isolation buffer (NIB: 20mM Tris-HCl, 50mM EDTA, 5mM Spermidine, 0.15mM Spermine, 0.1% mercaptoethanol, 40% Glycerol, pH 7.5) and followed the standard protocol for nuclei isolation.  Total RNA was extracted using a RNeasy plant kit (Qiagen catalog no. 74904). Approximately 40 μg total RNA was converted to cDNA using the mRNA-seq kit from Illumina. Construction of ChIP-seq libraries was according to the protocol of “preparing samples for ChIP sequencing of DNA” provided by Illumina.  Briefly, ChIPed DNA was end repaired using ‘End-It DNA End Repair Kit’ (Epicentre, ER0720).  The ‘dA’ base was then added to the 3’ ends of the blunt-ended DNA fragments using Klenow fragment (3’-5’exo-) (NEB, M0212S).  Linker for single read was ligated to the DNA molecules with ‘dA’ overhang using LigaFast kit (Promega, M8221).  After 2% agarose gel running with TAE buffer, the gel region containing about 150-300 bp DNA was excised for adapter ligated DNA size selection.  The adapter modified DNA fragments were enriched by PCR and purified by running 2% agarose gel in TAE buffer for isolation of DNA fragments in the range of 200-300 bp.  The purified DNA sample was ready for Illumina sequencing after quality validation. RNA-seq libraries were constructed using barcode method and then was sequenced by an Illumina Genome Analyzer II platform.