Discovery of Porcine MicroRNA by Solexa Sequencing and Large White pig) using standard t-test, which P-values were adjusted by using Benjamini and Hochberg’s method. Supporting Information Figure S1 Illustration of high-throughput identification of porcine microRNAs by deep sequencing. (TIF) Porcine microRNAs were identified by miRDeep Table S2 Putative novel porcine microRNAs which size range from 18–30 nt were identified by miRDeep program. (XLS) Stem-loop structures of putative novel microRNA with the PerfectHybTM buffer (Toyobo, Osaka, Japan) at 42uC. The blots were then washed with 26 SSC/0.1% SDS buffer three times at 42uC and then autoradiographed. The blots were visualized in a FLA 9000 biomolecular imager and analyzed with the Multi Gauge software (Fujifilm, Tokyo, Japan). Pig 5S rRNA served as the loading control. Stem-loop RT-PCR Real-time PCR was performed using a standard SYBR Green PCR kit (Toyobo, Japan) in the BioRad iQ5 Real-Time PCR Detection System according to the manufacturer’s instructions. Stem-loop RT primers were designed according to Chen et al (2005) and listed in the Table S17.The reverse transcriptase reaction using Stem-loop primers was performed according to the protocol of our previous studies[30]. New mammalian miRNA-array design In order to develop new tools to use the porcine miRNAs isolated through Solexa deep sequencing in biological studies, a new mammalian miRNA-array was designed according to the miRNAs submitted in miRBase and our porcine miRNAs. This miRNA-array contains 1551 specific miRNA probes, including 988 miRNAs of human, 350 miRNAs of rat, 627 miRNAs of mouse and 546 miRNAs of pig, respectively. U6 and tRNA probes were designed for internal positive control. Moreover, eight artificial probes of 30 nt named as Zip5, Zip13, Zip15, Zip21, Zip23, Zip25, Y2 and Y3 were used as external control. 50% DMSO were used as negative control. The probes were concatenated up to a length 40 nt (39 -end miRNA plus 59 -end PolyT) and attached to the slide surface Via a C6 59-amino- modifier. The probes were spotted to the 75 mm625 mm chip at a concentration of 40 mm using SmartArrayTM (CapitalBio Corp., Beijing, China) and the HEX labeled probes were used as positive control for quality of the spotting. Each probe was printed in triplicate on the miRNA-array. Porcine miRNAs expression detection MiRNAs expressed in liver were detected using our miRNA- array. Total liver RNAs from three Tongcheng pigs and three Large White pigs at about 25 kg body weight were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the low-molecular-weight RNA isolated using PEG reagent and labeled with Cy3 (Dharmacon, USA) according to the previous studies [48,49]. For slide hybridization, 5 mg Cy3 labeled RNA was dissolve in 20 ml hybridization mixture (15% formamide; 0.2%SDS; 36SSC; 506Denhardt’s) and hybridized overnight. Then, slides were washed in solution of 0.2% SDS, 26SSC at 42uC for 4 minutes, and in 0.26SSC for 4 minutes at room temperature. The slides were scanned with LuxScan 10K/A scanner (CapitalBio Company, China) and the raw pixel intensities were analyzed by LuxScan3.0 software (CapitalBio Company, China). The median pixel intensities that failed to exceed the negative control value by more than two standard deviations (Signal,negative mean+2 SD) were considered as negative hybridization. One miRNA was considered to be detected when it has positive signal at least at two miRNA-arrays. Statistical analysis of microarray data The normexp and quantile methods in limma package [50] were used to perform the background correction and normaliza- tion between arrays, respectively. After removing all control spots, 1594 miRNAs were fitted with linear model. Expression differences were compared between the two groups (Tongcheng PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16235 Table S1 program. (XLS) Table S3 precursors. (XLS) Table S7 program. (XLS) Table S4 Putative novel microRNAs location on the WGS sequence from NCBI database. (XLS) Table S5 Unique sequences with a perfect-match to known animal microRNA (using BLAST method search against miRBase 16.0). (XLS) Table S6 Unique sequences with not perfect-match to known animal microRNA (using BLAST method search against miRBase 16.0). (XLS) Porcine microRNAs were predicated by miRDeep Table S8 Conserved porcine microRNA on the WGS sequence from NCBI database. (XLS) Table S9 Chromosome location of putative novel and conserved microRNAs (Sanger database). (XLS) Table S10 isomiR_breakdown. (XLS) Table S11 Unique sequences with a perfect-match to known piwiRNA (using BLASTn method search against RNAdb2.0). (XLS) Table S12 MicroRNA sequences identified using the three different methods. (XLS) Table S13 Differentially expressed miRNAs in the liver tissue of Tongcheng pig and Large White. (XLS) Table S14 (XLS) Table S15 (XLS) Species of RNA and count number. Singleton vs. multiple sequence tags.