title: Sequencing of testis expressed small RNAs from the pig
design: Testis tissue was collected from mature boars (Sus scrofa domestica) and stored at -80°C. Total RNA was extracted directly from testis tissue using TRI Reagent® (Ambion®). Total RNA was applied to an urea-based denaturing polyacrylamide gel (10%) and run for 20 minutes (1200 V, 50 mA, 60W). The 20-35nt fraction was excised from the gel and resolved in water. We portioned the obtained sRNA sample into two fractions and conducted sodium periodate (NaIO4) treatment followed by ß-elimination with one of the two fractions according to the method applied by Behm-Ansmant and colleagues with minor adjustments regarding the sample volumes (Behm-Ansmant et al. 2011). A 5’-diphosphorylated and 3’-blocked RNA adapter (5’-rAppCTGTAGGCACCATCAATddC-3’, Integrated DNA Technologies®) was directionally ligated to the 3’ end of NaIO4 treated and untreated sRNA samples. For seperation of sRNA molecules linked to a 3’ adapter we conducted acid phenol chloroform (Life Technologies) extraction and ethanol precipitation followed by seperation of molecules ranging from 40 to 55 nt in length using polyacrylamide gel electrophoresis with subsequent gel extraction as described above. A second RNA adapter carrying an 4nt sequence tag and lacking a 5’-phospate was ligated to the 5' end of NaIO4 treated (5’-GACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3’) and untreated (5’-GACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGAUCGAA-3’) sRNA samples. The ligation reaction was stopped and RNA was purified by acid phenol chloroform extraction and ethanol precipitation and dissolved in water. Following cDNA synthesis using Superscript® III Reverse Transcriptase (Life Technologies), the sample was PCR amplified (forward primer for NaIO4 treated sample: 5’-ACATGGACTGAAGGAGTAGA-3’,  forward primer for untreated sample: 5’-ACATGGACTGAAGGAGATCG-3’, reverse primer for both samples: 5’-ATTGATGGTGCCTACAG-3’) and ethanol precipitated. Both tagged samples were high throughput sequenced in parallel on an Illumina HiSeq 2000 system.    Resulting constructs that are present in the library (in both orientations):    NaIO4 treated:  5'-ACATGGACTGAAGGAG-TAGA-[sRNA]-CTGTAGGCACCATCAAT-3'    Untreated:  5'-ACATGGACTGAAGGAG-ATCG-[sRNA]-CTGTAGGCACCATCAAT-3'