Total RNA was isolated from LV apex of 2-month-old WT and TtnΔC1-2 male (n = 6 each) mice using the RNeasy Fibrous Tissue Mini Kit (Qiagen). A Bioanalyzer (Agilent) was used to verify RNA quality and integrity and to determine concentration. Three samples per genotype were prepared with equal amounts of total RNA from two mice per sample. Library preparation and RNAseq were performed by the University of Chicago Genomics Facility following Illumina protocols for RiboZero depletion, TruSeq single-stranded total RNA library construction, and sequencing (2 × 100 bp paired-end reads) on the HiSEQ4000. The sequencing depth was sufficient to yield robust transcript level measurements with a per-sample mean of 40.0±0.5 × 106 paired-end reads aligned to the GRC38v11/mm10 Mus musculus reference genome. Analysis was performed on the Illumina BaseSpace cloud platform, the RNAExpress app was run which uses an analysis pipeline with the STAR short read aligner (which maps reads based on the GRC38/mm10 genome including identification of splice sites used)46 (link) which outputs counts for splice junction usage; counts for the titin region on chromosome 2 were extracted to produce Fig. 1b .
Basespace cloud platform
Manufactured by Illumina
BaseSpace cloud platform is a software product that provides a secure, cloud-based environment for data analysis, storage, and collaboration in life science research and clinical applications. It serves as a centralized platform to facilitate the management and processing of genomic data generated by Illumina's sequencing instruments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using basespace cloud platform
1
Titin Exon Deletion RNA-seq Analysis
2
Shotgun Sequencing and Taxonomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The Illumina BaseSpace cloud platform (https://basespace.illumina.com/) generated FASTQ files that were used for the analysis of the shotgun fragments. Sequence quality was ensured by trimming the reads using Trimmomatic 0.36 (Bolger, Lohse, & Usadel, 2014) , with the following parameters: CROP:147, HEADCROP:3, SLIDINGWINDOW:5:20, MINLEN:100.
All paired reads were aligned to the reference human genome (GRCh37-Hg19), using Bwa 0.7.15 (with option mem and parameters -M -t 20) (Li, 2013) . Sequences displaying a concordant alignment (a mate pair that aligns with the expected relative mate orientation and with the expected range of distances between mates) against the human genomes and all orphan reads were then removed from all subsequent analyses using SAMtools (Li et al., 2009) and awk custom filter.
Then, BLASTN 2.6.0 (Li et al., 2009; Altschul, Gish, Miller, Myers & Lipman, 1990; Camacho, 2013) was applied for aligning the remaining non-human reads to the NCBI (NR/)NT reference database. The alignments with an e-value lower than 1 × were further filtered to exclude matches with percentage of identity lower or equal than 95% and length lower than 100 bp.
Finally, MEGAN6 (Huson, Auch, Qi & Schuster 2007) with default parameters was used to perform the taxonomical analysis of the data, ranked for genus and species.
All paired reads were aligned to the reference human genome (GRCh37-Hg19), using Bwa 0.7.15 (with option mem and parameters -M -t 20) (Li, 2013) . Sequences displaying a concordant alignment (a mate pair that aligns with the expected relative mate orientation and with the expected range of distances between mates) against the human genomes and all orphan reads were then removed from all subsequent analyses using SAMtools (Li et al., 2009) and awk custom filter.
Then, BLASTN 2.6.0 (Li et al., 2009; Altschul, Gish, Miller, Myers & Lipman, 1990; Camacho, 2013) was applied for aligning the remaining non-human reads to the NCBI (NR/)NT reference database. The alignments with an e-value lower than 1 × were further filtered to exclude matches with percentage of identity lower or equal than 95% and length lower than 100 bp.
Finally, MEGAN6 (Huson, Auch, Qi & Schuster 2007) with default parameters was used to perform the taxonomical analysis of the data, ranked for genus and species.
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