GSM1160278: SmallRNA_B73_ear_I; Zea mays; RNA-Seq
All the samples were harvested and immediately frozen in liquid nitrogen and stored at -80°C. The total RNA from each sample was then isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. Small RNA library preparation and sequencing: the total RNAs from four developmental stages were pooled for Solexa sequencing. After small RNA cloning, the sequencing procedures were conducted as described previously. In brief, the sequencing was performed as follows: approximately 100 ug of total RNA was purified by polyacrylamide gel electrophoresis (PAGE), to enrich for molecules in the range of 18–30 nt, and ligated with proprietary adapters to the 5’ and 3’ termini of the RNA. The small RNA molecules samples were used as templates for cDNA synthesis. The cDNA was amplified with 18 PCR cycles and fragments of around 90 nt (small RNA + adaptors) were isolated from agarose gels. The purified DNA was used directly for cluster generation and Solexa sequencing analysis performed by the Illumina platform. The image files generated by the sequencer were then processed to produce digital-quality data. After masking of adaptor sequences and removal of contaminated reads, clean reads were processed for computational analysis. Degradome library construction: Small cDNA libraries using the sliced ends of polyadenylated transcripts from maize ears of four developmental stages were constructed according to previous report. In brief, poly(A) RNA was extracted from 200 μg of total RNA using the Oligotex kit (Qiagen), and it’s possessing 5’ monophosphates were ligated to an RNA adapter consisting of a MmeI recognition site in its 3’ end. After ligation, first-strand cDNA was generated using oligo d(T) and the PCR product was amplified using five PCR cycles. The PCR product was purified and digested with MmeI. The digested PCR product was then ligated to a double-stranded DNA oligo with degenerate nucleotides at the 5’ or 3’ end. The ligation product was further gel purified and amplified using 10 PCR cycles. The final PCR product was purified and sequenced using SBS sequencing technology.
GEO Accession
GSM1160278