Solexa sequencer

The sRNA libraries were constructed following a standard protocol (Illumina, USA). Small RNAs were purified from 10 μg of total RNA by polyacrylamide gel electrophoresis, and ligated first to a 5′ RNA adaptor and then to a 3′ RNA adaptor as previously described28 (link). Purified RNAs were reverse-transcribed to cDNA, followed by PCR amplification to generate the DNA pool16 (link). Five DNA pooled libraries were sequenced on an Illumina Solexa Sequencer at the Beijing Genomics Institute (BGI, http://www.genomics.cn/en/index) in Wuhan. Each sample was duplicated.
Five degradome libraries were constructed from cotton leaves to predict the potential target mRNAs following the methods described previously59 (link). Briefly, a 5′ RNA adapter with a MmeI recognition site at the 3′ end was ligated to the resulting 42 bp fragments consisting of a free phosphate at the 5′ end, followed by reverse-transcribed to cDNA. After PCR amplification, they were digested by the enzyme MmeI, and ligated to Illumina 3′ TruSeq adaptors, followed by PCR amplification with library-specific index primers and a common 5′ primer for multiplex sequencing. Five DNA pools were sequenced on an Illumina Solexa Sequencer at BGI.

The double-stranded cDNA was subjected to library preparation using the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA), through a four-step protocol including end repairing, addition of adenylate 3′ ends, adapter ligation, and cDNA template enrichment. The amplification program were: 98 °C 30 s; 98 °C 10 s, 60 °C 30 s, 72 °C 30 s for 15 cycles; 72 °C for 5 min, and hold at 4 °C. To determine the quality of the libraries, a QubitW 2.0 Fluorometer and Qubit™ dsDNA HS (Invitrogen, Grand Island, NY) were first used to determine the DNA concentration of the libraries, and a FlashGel DNA Cassette (Lonza, USA) or Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA) was used to determine the product size of the libraries, with good libraries typically 250–300 bp. The product was used directly for cluster generation using an Illumina Solexa Sequencer according to the manufacturer’s instructions.
RNA 2 × 100 bp paired-end sequencing was performed using the standard protocol for the Illumina Genome Analyzer IIx. The cDNA library for each sample was loaded onto a single lane of an Illumina flow cell. The image deconvolution and calculation of quality values were performed using a Goat module (Firecrest v.1.4.0 and Bustard v.1.4.0 programs) with Illumina pipeline v.1.4. Sequenced reads were generated by base calling using the Illumina standard pipeline.

Equal amounts of frozen Mg-deficient or -sufficient leaves from five plants (one plant per pot) were mixed as a biological replicate. Total RNA was extracted from 0.1 g mixed frozen samples using TRIzol reagent (Invitrogen, Carlsbad, CA) following manufacturer's instructions. Mg-deficient and -sufficient leaf sRNA libraries were constructed according to Lu et al. (2014 (link)). Illumina sequencing was performed on a Solexa sequencer at the Beijing Genomics Institute (BGI), Shenzhen, China (Lu et al., 2014 (link)).