Computational protocol: Identification of TERRA locus unveils a telomere protection role through association to nearly all chromosomes

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Protocol publication

[…] Measure of 150 ng of RNA was randomly fragmented, converted to double-stranded cDNA and subsequently processed through enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's ‘Preparing Samples for Sequencing of mRNA’ manual (Part# 1004898 Rev. A). A fraction of 175–225 bp was extracted from an agarose gel and adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers (15 cycles; Illumina). The resulting purified cDNA library was applied to an Illumina flow cell (Illumina) for cluster generation and sequenced on the Genome Analyzer II. The obtained reads were all single-end 36 bp. Primary data analysis was performed with GAPipeline-1.4 (Illumina) generating FastQ files. The single-end reads were aligned to the reference genome Mus genome assembly mm10 (GRCm38) with TopHat v2.0.4 (ref. ), using Bowtie 0.12.7 (ref. ) and Samtools 0.1.16 (ref. ) allowing up to two initial read mismatches, and up to 20 mappings when multiple alignments are possible (based on alignment scores). Transcripts assembly, estimation of their abundances and differential expression were calculated with Cufflinks 1.3.0 (ref. ), using the mouse genome annotation data set GRCm38/mm10 from the UCSC Genome Browser. The expression value of each gene between the conditions is shown as the log10 of the FPKM fold-change (FPKM: fragments per kilobase of transcript per million fragments mapped). Raw data can be access at http://www.ncbi.nlm.nih.gov/sra/ with the identifier SRA059274. Custom CAGE tag track was prepared using the Fantom3 collection of CAGEtag starting sites. The link to UCSC session with custom ‘CAGE tag’ track is: http://genome.ucsc.edu/cgi-bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=ilsilanes&hgS_otherUserSessionName=CAGE%20tag. […]

Pipeline specifications

Software tools TopHat, Bowtie, SAMtools, Cufflinks
Databases FANTOM UCSC Genome Browser
Application RNA-seq analysis
Organisms Mus musculus