Growth of the algae
Chlamydomonas reinhardtii CC849 were obtained from Chlamydomonas Genetic Centre (c/o Dr. Elizabeth H. Harris, Department of Botany, Duke University, Durham, NC27706, USA). The algal strain was grown in a Tris-Acetate-Phosphate (TAP) medium at 25°C and under continuous cool-white fluorescent lamps(≈200 μmol photons m-2 s-1). To impose S deprivation [6, 8], the liquid cultures were grown into mid-logarithmic phase, algal cells were collected by centrifugation, were washed twice with liquid TAP medium without S (TAP-S, for 1 L of Medium: 2X Filner's Beijernicks Solution 25 ml; 1 M Potassium Phosphate 1 ml; Trace mineral solution 1 ml; Tris-Base 2.42 g; adjust pH to 7.0 by Glacial Acetic Acid. Sulfur-deprivation media (TAP-S) were prepared by replacement of the S-salts by their chloride counterparts). Equal numbers of cells were resuspended in TAP or TAP-S under continuous illumination for up to 72 h, with cell aliquots were collected for RNA isolation, and the sulfate concentration in the supernatant was determined by Dionex ICS-1100 ion chromatogram. Small RNA library construction was carried out as follows: for the + S library, RNA was isolated from the algal cells which were resuspended in sulfur-replete TAP media; for the -S library, RNA was isolated from the algal cells which were resuspended in sulfur-free media TAP-S.
Preparation of total RNA
Total RNA was extracted using Trizol reagent (Invitrogen). The 72 h cells cultured at 25°C in TAP and TAP-S were collected. Total RNA was extracted according to the manufacturer's protocol. The quality of RNA was examined by using an Agilent 2100 Bioanalyzer. The same amount of total RNA was used to construct the two libraries and the samples were prepared in a similar manner. Sequencing of the two libraries was performed on the Illumina's Solexa Sequencer and the samples were run side by side.
Small RNA library construction and high-throughput sequencing
After PAGE purification of small RNA molecules under 30 bases and ligation of a pair of Solexa adaptors to their 5'and 3'ends (Illumina, San Diego, CA. USA), the small RNA molecules were amplified using the adaptor primers for 17 cycles and the fragments around 90 bp (small RNA + adaptors) were isolated with agarose gel. The purified DNA was used directly for cluster generation and sequencing analysis using the Illumina's Solexa Sequencer according to the manufacturer's instructions. The image files generated by the sequencer were processed to produce digital-quality data. The following procedures were performed with Solexa to summarize the data: evaluation of the sequencing quality, calculation of the length distribution of small RNA reads, and filtration of the reads contaminated by rRNA, tRNA, mRNA, snRNA, and snoRNA. Finally, clean reads were compared with a miRBase database (release 15.0).
Data quality and length distribution
We eliminated some contaminant reads from the fq file and to obtain the final clean reads and then summarized the length distribution of these clean reads. Normally, length of small RNA is between 18 nt and 30 nt. The length distribution analysis was helpful to see the composition of small RNA samples. For example, miRNA is normally 21 nt or 22 nt, siRNA is 24 nt, and piRNA is 30 nt. The data was processed by the following steps: 1) Elimination of low quality reads (the criteria for this was listed in the explanation of meaning of each row in the result tables); 2) Elimination of reads with 5' primer contaminants; 3) Elimination of reads without 3' primer; 4) Elimination of reads without the insert tag; 5) Elimination of reads with poly A; 6) Elimination of reads shorter than 18 nt; 7) Summarization of the length distribution of the clean reads.
Mapping to genome
We mapped the small RNA tags to the genome by SOAP to analyze their expression and distribution on the genome. Program and Parameters: soap -v 0 -r 2 -s 7 -p 7 -a clean.fa -d ref_genome.fa -o match_genome.soap.
Summary of known miRNA alignment
We aligned the small RNA to the miRNA precursor of corresponding species (using mature miRNA if there was no precursor information of that species in miRBase14.0) to obtain the miRNA count as well as base bias on the first position of identified miRNAs with certain lengths and on each position of all identified miRNAs respectively.
Small interfering RNA (siRNA) is a 22-24 nt long double-strand RNA, each strand of which is 2 nt longer than the other on the 3' end. According to this structural feature, we aligned tags from clean reads to each other to find sRNAs meeting this criteria. These tags might be potential siRNA candidates. Program and Parameters: Software developed by BGI-tag2siRNA.
Alignment to Genbank
We annotated the small RNA tags with rRNA, scRNA, snoRNA, snRNA and tRNA from Genbank and elimination of matched tags from unannotated tags. Program and Parameters: blastall -p blastn -FF -e 0.01
Alignment to Rfam
Annotation of the small RNA tags with sequences from Rfam and elimination of matched tags from unannotated tags. Program and Parameters: blastall -p blastn -FF -e 0.01
Small RNA annotation
We Summarized all prior alignments and annotation before. In the previous alignment and annotation, some small RNA tags may be mapped to more than one category. To make every unique small RNA map to only one annotation, we followed the following priority rule: rRNAetc(in which Genbank > Rfam) > known miRNA > repeat > exon > intron. Program and Parameters: Software developed by BGI-tag2annotation
Known miRNA expression profile
We aligned small RNA tags to the miRNA precursor/mature miRNA of corresponding species in miRBase14.0. To show detailed information of alignment, including structure of known miRNA precursor, length and count of tags from the sample, etc, click the miRNA id in the left table to see detailed information of that miRNA. Note: Only part of the known miRNA alignment are shown in this report.
Novel miRNA prediction
The characteristic hairpin structure of miRNA precursor was used to predict novel miRNA. We used the predictive software Mireap to predict novel miRNA by exploring the secondary structure, and the minimum free energy of the unannotated small RNA tags which could be mapped to genome. Mireap can be accessed from the following link: http://sourceforge.net/projects/mireap/
Program and parameters
Software developed by BGI- Mireap Minimal miRNA sequence length (18); Maximal miRNA sequence length (25); Minimal miRNA reference sequence length (20); Maximal miRNA reference sequence length (23); Maximal copy number of miRNAs on reference (20); Maximal free energy allowed for a miRNA precursor (-18 kcal/mol); Maximal space between miRNA and miRNA* (300); Minimal base pairs of miRNA and miRNA* (16); Maximal bulge of miRNA and miRNA* (4); Maximal asymmetry of miRNA/miRNA* duplex (4); Flank sequence length of miRNA precursor (20); ram and Parameters: blastall -p blastn -FF -e 0.01.
Differential expression of known miRNA
Comparison of the known miRNA expression between two samples to determine the differentially expressed miRNA. The procedures are shown as below: (1) Normalize the expression of miRNA in two samples (control and treatment) to get the expression of transcript per million(TPM). Normalization forum:Normalized expression = Actual miRNA count/Total count of clean reads*1000000; (2) Calculate fold-change and P-value from the normalized expression according the Bayesian method developed by Audic and Claverie (1997) . Then generate the log2 ratio plot and scatter plot.
Fold-change forum: Fold change = log 2 (treatment/control)
The N1 and x represent total counts of clean reads and normalized expression level of a given miRNA in sRNA library of + S sample, respectively. The N2 and y represent total counts of clean reads and normalized expression levels of a given miRNA in sRNA library of -S sample, respectively.
Quantitative RT-PCR validation of miRNAs
Sulfur-deprivation procedure is the same as previously described. Both total RNA from 72 h cells cultivated in TAP and TAP-S were isolated respectively for quantitative RT-PCR using the TRIZOL reagent (Invitrogen, Life Technologies, Carlsbad, CA). Low molecular weight RNA and high molecular weight RNA were separated with 4 M LiCl. sRNAs were polyadenylated at 37°C for 60 min in a 50 μl reaction mixture with 1.5 μg of total RNA, 1 mM ATP, 2.5 mM MgCl2, and 4 U poly(A) polymerase (Takara, Japan). Poly (A)-tailed sRNA was recovered by phenol/chloroform extraction and ethanol precipitation. The sRNAs were dissolved, treated with RNase-free DnaseI (Takara, Japan) and reversely transcribed using poly (T) adapter. Real-time PCR was performed using SYBRR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) and all the primers used were as listed in Additional file 2: Table S1. For each reaction, 1 μL of diluted cDNA (equivalent to 100 pg of total RNA) was mixed with 10 μL of 2 × SYBR green reaction mix (SYBRR Green qRT-PCR Master Mix; Takara, Japan), and 5 pmol of the forward and the reverse primers were added to make a final volume of 20 μL. The conditions for the PCR amplification were as follows: polymerase activation was conducted at 95°C for 30 s; followed by 40 cycles at 95°C for 5 s, 60°C for 31 s. The specificity of the primer amplicons was tested by analysis of a melting curve. The U4 snoRNA was used as a reference gene in the real-time PCR detection of miRNAs. The data was analyzed using the 2--ΔΔCt program, all with an R2 above 0.998. For this quantitative RT-PCR analysis, 3 technical replicates and 2 biological replicates were used.