Nine-day-old seedlings were acclimated for five days by opening the plastic cap and adding tap water onto the MS agar medium. Five days later, 2-week-old seedlings were moved to tap water in a glass tube and used for stress treatments and controls. For drought treatment, seedlings were air-dried for 8 h. For salt stress treatment, seedlings were transferred to 300 mM NaCl solution for 8 h. For cold stress treatment, seedlings were transferred to 4° C in a cold room under continuous light for 8 h. For heat stress treatment, seedlings were moved to 42°C for 8 h. Dual stresses were also treated under the same conditions. Control plants were sampled at the same time. All the collected tissues were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. For nutrient starvation treatments, the endosperm was first removed from 9-day-old seedlings to avoid nutrient transport. Seedlings were then transferred to MS nutrient-deficient media or MS media (for control). For MS nutrient-deficient media, specific components of MS were omitted or substituted with others as follows: For nitrogen-deficient medium, NH4NO3 was omitted and KCl was substituted for KNO3. For phosphate-deficient medium, KH2PO4 was omitted. For potassium-deficient medium, KNO3 was omitted. For sulfate-deficient medium, all SO4 was substituted with Cl2. After five days of treatment, shoots and roots were separated for independent analysis. Mature plants at the booting stage were treated with various environmental stresses under the same conditions as described above, but for three days with the exception of the drought treatment. For drought treatment, pots were removed from the water, and holes were punched in the pots to help draining, and dehydrated for seven days. After stress treatment, mature panicles were sampled, which were about 15 cm and wrapped with panicle sheath.
Growth protocol
Rice seeds (Oryza sativa L, ssp. japonica cv. Nipponbare) were husked, surface-sterilized with 3% sodium hydrochlorite, washed with sterile water three times, and germinated on half-strength Murashige and Skoog (MS) medium containing 0.2% (w/v) Phytagel in a sterile plastic box. Plants were grown in a growth chamber under conditions of 12 h light at 28°C and 12 h dark at 25°C.
Extracted molecule
total RNA
Extraction protocol
Small RNA libraries were constructed as previously described (Lu et al. 2007) and sequenced on llumina 1G or IIGX analyzer. Sequencing was performed at Illumina, National Center for Genome Research, Cold Spring Harbor Laboratory, or the Delaware Biotechnology Institute.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
Illumina Genome Analyzer II
Description
RK2
Data processing
The adaptor sequences were identified and removed by using a Perl script. Small RNAs shorter than 15 nt were removed. Data matrix contains distinct small RNAs with their raw abundance after removing the adapter sequences.
