GSM2650257: clsy1-7,4-1_rep1_smRNA-seq; Arabidopsis thaliana; ncRNA-Seq
4 un-opened flower buds (stage 12 and younger) from individual mutant plants as well as 3 individual wild-type (WT) controls were collected, frozen in liquid nitrogen and kept at -80°C until use. Total RNAs were isolated using Trizol reagent (Life Tech, Cat# 15596018) following the manual and dissolved in 250μL RNase-free water. These total RNAs then were enriched for small RNAs through a polyethylene glycol (PEG) precipitation step. One volume of a 20% PEG 8000/2M NaCl solution was added to each RNA sample, mixed well and kept on ice for more than 30 minutes (min) and then centrifuged at 13,000rpm for 15min at 4°C. The supernatant, containing the small RNAs, was collected and precipitated with 1 volume of cold isopropanol, mixed and incubated on ice for 30min. Then the small RNA samples were recovered by centrifuged at 13,000rpm for 30min at 4°C. The resulting small RNA samples were run on 15% polyacrylamide-8M urea gels and performed size-selection of fragments from 17-29 nucleotide (nt) following the ZR-small RNA ladder (zymo research, Cat# R1090). The small RNAs in the gels were recovered by incubating with 0.3M NaCl overnight, filtering the gels and add 2.5 volumes of 100% ethanol and 3μL of glycogen for precipitation over 2 hours (h) at -80°C. The resulting small RNAs were used for library preparation using NEBnext Multiplex Small RNA Library Prep Set for Illumina (Cat# E7300) following the manual. The final library products were further purified by running 8% polyacrylamide gels and performed size-selection from 140-160nt as described above. The purified libraries were equally pooled and submitted for single-end 50 bp (SE50) sequencing on HiSeq 2500 (Illumina) at the Next Generation Sequencing Core of the Salk Institute for Biological Studies.
GEO Accession
GSM2650257