The oligonucleotide ligation was carried out by using the total RNA and a RNA/DNA chimeric 5' oligonucleotide (5' phosphorylated, 3' RNA) containing an EcoRI recognition site (sequence: 5' acg ctc aca gaa ttc AAA 3', the upper-case indicates the RNA part of the oligonucleotide, lower-case indicates the DNA part of the oligonucleotide) and a specific 3' oligonucleotide, containing another EcoRI recognition site (sequence: 5'(phosphate)-UNN nn gaattc tca cga ggc cag cgt-(biotin) 3' NNnn sequences are means barcode tags to distinguish each library. The upper-case indicates the RNA part of the oligonucleotide, lower-case indicates the DNA part of the oligonucleotide).<br />Ligated short RNAs were separated from 5'/3'adaptor dimers on a polyacrylamide denaturing gel. 37~270 nt short RNAs, running above adaptor dimers, were excised and eluted from the gel in elution buffer. The cDNA synthesis was carried out from the purified short RNAs using 3'-RT-PCR primer (sequence: 5'-gactagctggaattcgcggttaaa-3') and MMLV reverse transcriptase RNaseH minus (Promega). The cDNA products derived from short RNAs were amplified by PCR using adaptor-specific primers: Primer 1 (shortRNA3'RT-PCRprimer): 5'(biotin)-gca cgc tgg cct cgt gag aat tc-3'; Primer 2 (shortRNA5'PCRprimer): 5'(biotin)-cag cca acg ctc aca gaa ttc aaa-3'. The PCR products were purified from a polyacrylamide gel. The each broad bands corresponding to cDNAs were gel-purified.<br />After a second cycle of PCR, the products were digested with EcoRI, followed by purification of the insert and removal of remaining fraction corresponding to the digested linker, by 12% native TAE polyacrylamide electrophoresis. After the elution from gel, the cDNAs were concatenated for over night at 15°C with T4-DNA ligase. Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - were added to each fragment, and purify with GFX column.
<HTML><HEAD><TITLE>SHORT_RNA</TITLE><meta http-equiv="content-type" content="text/html; charset=UTF-8"/><script type="text/javascript" src="/sw/js/panel/panelDescription.js"></script></HEAD><BODY>"The oligonucleotide ligation was carried out by using the total RNA and a RNA/DNA chimeric 5' oligonucleotide (5' phosphorylated, 3' RNA) containing an EcoRI recognition site (sequence: 5' acg ctc aca gaa ttc AAA 3', the upper-case indicates the RNA part of the oligonucleotide, lower-case indicates the DNA part of the oligonucleotide) and a specific 3' oligonucleotide, containing another EcoRI recognition site (sequence: 5'(phosphate)-UNN nn gaattc tca cga ggc cag cgt-(biotin) 3' NNnn sequences are means barcode tags to distinguish each library. The upper-case indicates the RNA part of the oligonucleotide, lower-case indicates the DNA part of the oligonucleotide).<br />Ligated short RNAs were separated from 5'/3'adaptor dimers on a polyacrylamide denaturing gel. 37~270 nt short RNAs, running above adaptor dimers, were excised and eluted from the gel in elution buffer. The cDNA synthesis was carried out from the purified short RNAs using 3'-RT-PCR primer (sequence: 5'-gactagctggaattcgcggttaaa-3') and MMLV reverse transcriptase RNaseH minus (Promega). The cDNA products derived from short RNAs were amplified by PCR using adaptor-specific primers: Primer 1 (shortRNA3'RT-PCRprimer): 5'(biotin)-gca cgc tgg cct cgt gag aat tc-3'; Primer 2 (shortRNA5'PCRprimer): 5'(biotin)-cag cca acg ctc aca gaa ttc aaa-3'. The PCR products were purified from a polyacrylamide gel. The each broad bands corresponding to cDNAs were gel-purified.<br />After a second cycle of PCR, the products were digested with EcoRI, followed by purification of the insert and removal of remaining fraction corresponding to the digested linker, by 12% native TAE polyacrylamide electrophoresis. After the elution from gel, the cDNAs were concatenated for over night at 15°C with T4-DNA ligase. Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - were added to each fragment, and purify with GFX column."</BODY></HTML>
SHORT_RNA