Alexandra Ledermann 6 No Dvd 🖐🏿
Alexandra Ledermann 6 No Dvd 🖐🏿
Alexandra Ledermann 6 No Dvd
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A total of 2 µg of DNA was incubated at room temperature with 100 pmol of each DNA sequence, 10 mM sodium phosphate (pH 7.0) and 20 mM MgCl~2~ (as indicated) for 1 h, and the reaction was stopped by incubation with 0.1 mg of proteinase K for 30 min at 37°C. The biotin-labeled oligo(G) DNA was then purified and fragmented in a hydrolysis solution containing 10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl~2~, and 1 mM ZnCl~2~. The DNA was purified by using the ZymoPURE kit (Zymo Research) and subsequently subjected to miRNA sequencing. The oligo(G) DNA was subjected to RNA sequencing in the same manner as described previously.
Data Processing {#s4g}
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The 3′-tag sequence for the adaptor was trimmed from each read, and the reads were aligned against a customized reference database (which consisted of mouse miRNA sequences from Sanger miRBase, release 13.0) using the Bowtie aligner 2.0.6 with the following parameters: \–no-unal, \–no-mixed, \–n-best 100, and \–best. Sequences aligned to the mouse genome were subsequently filtered to remove reads mapped to the exon or intron regions of the mouse genome. Sequences aligned to rRNA, tRNA and other non-coding RNA were also removed. miRNA expression abundance was normalized to the total number of miRNA reads per library.
Supporting Information {#s5}
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Alexandra Ledermann 6 No Dvd
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