For RNA-seq from mESCs, total RNA was extracted from one well of a six-well plate of 60%–80% confluent cells with QIAzol reagent following the manufacturer's recommendation. Libraries were prepared with NEBNext Ultra II Directional RNA Library Kit for Illumina with prior use of NEBNext rRNA Depletion Kit v2 (E7405, NEB) following the manufacturer's protocol. Libraries were amplified using eight PCR cycles and sequenced on a NextSeq 500 (Illumina) in 75 bp single-end read mode.
For downstream analyses, adaptor sequences were removed from the reads with cutadapt (Kechin et al. 2017 (link)) (1.18) using default settings. For the analysis of differentially expressed retrotransposons, consensus sequences of rodent retrotransposons were retrieved from Repbase (24.01) and used to map the processed reads using bowtie2 (Langmead and Salzberg 2012 (link)) (2.4.2) with default settings. The number of mapped reads per retrotransposon were counted and analyzed using DESeq2 (1.32.0) (Love et al. 2014 (link)).
For downstream analyses, adaptor sequences were removed from the reads with cutadapt (Kechin et al. 2017 (link)) (1.18) using default settings. For the analysis of differentially expressed retrotransposons, consensus sequences of rodent retrotransposons were retrieved from Repbase (24.01) and used to map the processed reads using bowtie2 (Langmead and Salzberg 2012 (link)) (2.4.2) with default settings. The number of mapped reads per retrotransposon were counted and analyzed using DESeq2 (1.32.0) (Love et al. 2014 (link)).
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