title: rice ssRNA-seq
design: rice ssRNA-seq
library_construction_protocol: Oryza sativa cv. Nipponbare seeds were used in all experiments.  Seeds germinated at 28  C in dark for 2 days were transferred to plant growth chamber to grow for 14 days under controlled conditions (12 h light 30  C/12h dark 24  C cycles) to harvest the seedlings. For stress treatments, 14-day-old seedlings were incubated in solutions containing 20% PEG-6000, 200 mM NaCl, for 4 h at 30 C, respectively. For cold treatment seedlings at the same developmental stage were treated at 4 C for 24 h in dark. Total genomic DNA was extracted from leaf tissues using DNase (New England Biolabs), and total RNA was isolated using the TRIzol reagent (Invitrogen).  The OligoTex mRNA midi kit (Qiagen) was used to purify poly(A) mRNA from the total RNA samples.  Next, mRNAs were fragmented by the RNA fragmentation kit (Ambion).  The first cDNA strand was synthesized using random hexamer primers and the second strand cDNA was synthesized where dUTP was used instead of dTTP.  Then the dUTP was digested. Small RNAs, ranging from 18-35 bp were enriched by polyacrylamide gel electrophoresis, and ligated to 5' and 3' adapters. The ligation product was reverse-transcript into cDNA, which was then amplified by 15 PCR cycles.
attributes.tag: Experimental Factor: GROWTHCONDITION
attributes.value: cold stress