GSM5718040: clsy1-7_2-2_3-1_4-1_Ov_rep2_smRNA-seq; Arabidopsis thaliana; ncRNA-Seq
Total RNAs were isolated using Trizol reagent (Life Tech, Cat# 15596018) following the manual and dissolved in 250μL RNase-free water. 2.0 µg of total RNA from Ar and Lv tissues, or ~0.5ug of total RNA from Ov, were used for smRNA enrichment 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 small RNAs were used for library preparation with the NEBnext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Cat# E7300) following the user's manual. The final library products were further purified using an 8% polyacrylamide gel to excise 130-160nt products relative to the pBR322 DNA-MspI Digest ladder (New England Biolabs, Cat# E7323AA). The purified libraries were pooled and sequenced (single end 50 bp, SE50) on a HiSeq 2500 machine (Illumina).
GEO Accession
GSM5718040