Preparation of Small RNA Libraries for High-Throughput Sequencing

C. Malone, J. Brennecke, B. Czech, A. Aravin, G. J. Hannon
2012 Cold Spring Harbor Protocols  
This protocol details the process of small RNA cloning for sequencing on the Illumina/Solexa sequencing platform, but it can be easily modified for use on other next-generation platforms (e.g., SOLiD, 454). This procedure is designed to clone canonical small RNA molecules with 5 ′ -monophosphate and 3 ′ -hydroxyl termini. Modifications, such as the presence of a 2 ′ -O-methyl group, can reduce efficiency, although not sufficiently to negate the utility of the approach. Other termini
more » ... termini modifications, such as a 5 ′ triphosphate or a 3 ′ phosphate, can be altered by enzymatic treatment before cloning. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Bovine serum albumin (BSA; 100×) Chloroform Decade Marker (Ambion) Diethyl pyrocarbonate (DEPC)-Milli-Q H 2 O Dimethylsulfoxide (DMSO) Ethanol (100%, 70%) Ethidium bromide [γ-32 P]ATP (Perkin Elmer) Gel Loading Buffer II (1×) (Ambion) Gel-loading dye (6×) (Fisher) GeneRuler 50-bp DNA ladder (Crystalgen) GlycoBlue (Ambion) NaCl (0.4 M) Adapted from RNA: A Laboratory Manual,
doi:10.1101/pdb.prot071431 pmid:23028068 fatcat:udvq7qqf25c37pb6fp56b26tte