Profiling Small RNA Transcripts from Plant Roots

About 0.1 g mixed frozen B-sufficient and -deficient roots from five replictations were used to extract RNA. Total RNA was extracted from frozen roots using TRIzol reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. Two sRNA libraries were constructed according to Wang et al. [62 (link)]. Briefly, sRNAs were isolated from the total RNA by size fractionation with 15% Tris-borate-EDTA urea polyacrylamide gel (TBU). Then the sRNAs were ligated with 5' and 3' adaptor by T4 RNA ligase after being dephosphorylated by alkaline phosphatase. The adaptor-ligated sRNAs were transcribed to single-stranded cDNA using Superscript II reverse transcriptase (Invitrogen). Thereafter, the single-stranded cDNA was used as templates for double-stranded synthesis by PCR amplification using the primer designed according to the adapter sequence. The obtained PCR products were sequenced on a Solexa sequencer (Illumina) at the Beijing Genomics Institute (BGI), Shenzhen, China.