GSM2527192: C_III_3; Zea mays; RNA-Seq
Total RNA was isolated from the first three10-mm leaf segments from the basal 30 mm of leaf 4 three days after leaf emergement, during steady-state growth, from each treatment. The TRIzol reagent (Invitrogen) was used as described in the manufacturer’s protocol. Then, 0.5 to 1.0 mg of total RNA was incubated with RNase-free DNase I (1 unit/mL) following the protocol provided by the manufacturer to remove possible genomic DNA. Prior to library preparation the RNA quality and integrity was assessed by using a gel cartridge on a QIAxcel platform (Qiagen, Hilden, Germany). Library preparation was done using the TruSeq® Stranded mRNA sample preparation 96 rcx kit (Illumina™) following the low sample protocol according to Illumina™ guidelines. Briefly, approximately 2.5 µg of total RNA was diluted and purified using RNA purification beads targeting the poly-A tail of the mRNA and subsequently was fragmented by means of the enzymes provided in the kit. After the cDNA synthesis adenylation of 3’ ends and ligation of the adaptors were employed. Adaptors were ligated in 12-plex formations, allowing the pooling of 12 samples after the PCR enrichment of the library. Subsequently, the library was quantified using PicoGreen® dye (Life Technologies™) as described in the manufacturer’s protocol. In order to accurately quantify the concentration in nM of our pools, the Kapa SYBR® FAST universal qPCR kit (Kapa Biosystems™) for Illumina™ sequencing was used to quantify the number of the amplifiable molecules in the pools and the Bioanalyzer® (Agilent Technologies™) to determine the average fragment size of our pools. These measurements allowed optimizing the flow cell clustering and proceed with the Run. The illumina HiSeq 1500 was used in the high throughput mode together with the TruSeq® SBS Kit to sequence the samples in a 25 cycle pair-end run. Following the analysis of these runs, rapid run was performed on the samples having yielded below 5 million reads. The rapid runs were performed on the same pools as in the first sequencing round. Data from these rapid runs were added to the high throughput data. Groups of 12 samples were pooled at equal concentrations to create eight pools for each of the lanes of the flow cell
GEO Accession
GSM2527192