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Cloning and sequencing of chicken miRNAs. Secondary CEFs, prepared by routine techniques, were infected with the RB1B strain of MDV for 24 h at a multiplicity of infection of 25,000 PFU/106 cells. Protocols developed previously in one of our laboratories were used to construct the libraries (49). Briefly, RNA was isolated using TRIzol and size fractionated using polyethylene glycol (PEG) precipitation. The low-molecular-weight fraction was electrophoresed ona 15% polyacrylamide–8 M urea gel, and small RNAs (20 to 27 nt) were extracted from the gel and purified. Both 5 and 3 RNA adapters (Dharmacon Research, Boulder, CO) were sequentially ligated onto the RNA using T4 RNA ligase (Ambion, Austin, TX). The 5 RNA adapter (5-OH-GGUCUUAGUCG CAUCCUGUAGAUGGAUC-OH 3) and 3 RNA adapter (5-P-CACUGAU GCUGACACCUGC-idT-3; idT is inverted deoxythymidine) were designed to prevent self-ligation, and the ligation products were purified following each step. The RNA was then reverse transcribed (Superscript reverse transcriptase [RT]; Invitrogen) using a primer complementary to the 3 adapter. cDNA inserts were amplified by PCR using primers corresponding to both adapters. Amplicons were sequenced by using the 454 Life Sciences system that utilizes pyrosequencingbased, sequence-by-synthesis, high-throughput, parallel sequencing (52). Sequence data were filtered for adapter sequences and clustered (allowing a 4-base overhang or mismatch at either end), and the insert sequence was analyzed by comparing it to the chicken and MDV genomes and to the miRNA database (55) using BLAST (3). |
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