Prior to alignment, reads were demultiplexed and read fragments were trimmed for adaptors and for quality using Cutadapt (version 1.9.1;(Martin, 2011)). Reads were eliminated if the trimmed length of either read fragment was <20 nucleotides. Next, bowtie2(v 2.2.6) (4) was used to align reads to ribosomal RNA, and the unmapped reads were retained. These will be referred to as “cleaned” reads. The RNA-Seq Expectation Maximization (RSEM v1.2.31) algorithm (Li and Dewey 2011) was used to estimate the number of aligned reads for individual Ensembl transcripts (version 96). The number of reads aligned to each Ensembl gene was calculated as the sum of the number of reads aligned to individual Ensembl transcripts annotated to that gene.
+ +Prior to normalization and transformation, a detection above background filter was applied where we required a gene to have at least 2/3 of the samples have 1 count or more to be considered expressed above background. All genes that did not pass this detection above background criteria were removed. Count values were normalized using RUVg (PMID: 25150836) and transformed using the regularized log function (PMID: 25516281).
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