GSM2586400: C1; Zea mays; miRNA-Seq
Total RNA was isolated separately from each sample (IE, immature embryos; C1, one month; C4, four months and C10, ten months subcultured callus) using the TRIzol reagent (Thermofischer Scientific) according to the manufacturer’s instructions. For sRNA purification, the step of 70% ethanol wash was substituted by 96% ethanol wash. The quality of the total RNA was tested by 1% agarose-gel electrophoresis and the concentration was measured using the Agilent 2100 Bioanalyzer. Small RNAs ranging between 18–30 nt were isolated from total RNA on 15% TBE-urea denaturing polyacrylamide gel, and then ligated to a 3' DNA adaptor (IE and C4 libraries: CTGTAGGCACCATCAAT; C1 and C10 libraries: ATCTCGTATGCCGTCTTCTGCTTG). Then, ligated to a 5' RNA adaptor (GUUCAGAGUUCUACAGUCCGACGAUC). It was reverse-transcribed using the RT primer (IE and C4 libraries: CAAGCAGAAGACGGCATACGAATTGATGGTGCCTACAG; C1 and C10 libraries: CAAGCAGAAGACGGCATACGA). Subsequently, a PCR reaction was performed using. The forward primer was (AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) and as the reverse primer (CAAGCAGAAGACGGCATACGA). The amplified cDNA constructs were purified and mixed as follows: (IE + C1) and (C4 + C10) for multiplexed sequencing by UUSMD (UNAM-IBT, Mexico) using the Genome Analyzer IIx from Illumina.
GEO Accession
GSM2586400