mRNA was purified from 1μg of total RNA extracted as above using a NEBNext Poly(A) mRNA Magnetic isolation module (New England Biolabs). First and second strand cDNA was synthesized using NEBNext synthesis modules (New England Biolabs), purified using Agencourt AMPure XP beads (Beckman Coulter), and quantified using Quant-iT PicoGreen dsDNA assay (Life Technologies). Libraries were prepared from 10ng ds-cDNA using an Ovation SP ultralow library system (Nugen) in combination with a Mondrian SP work station (Nugen). Libraries were amplified by PCR for 15 cycles, quantified, denatured and 10pmol of each library were pooled and sequenced on a 1x50bp program on a Hiseq 2000 or 2500 (Illumina). After sequencing, the BCL file was demulitiplexed and converted to a FASTQ file using CASAVA (Illumina). Files were aligned to the mm10 UCSC genome using TopHat splice junction mapper with Bowtie2 [21 (link)]. Features were counted and a count table produced using Rsubread[22 (link)]. Raw and processed data including batch information and output from differential expression analysis with DESeq2 is available in the GEO repository (https://www.ncbi.nlm.nih.gov/geo ) under series record GSE98492.
Protocol detail
RNA-seq library preparation and sequencing
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Corresponding Organization : National Institutes of Health
Other organizations : University of Southampton, University of Cambridge
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Variable analysis
independent variables
- MRNA purification from 1μg of total RNA using a NEBNext Poly(A) mRNA Magnetic isolation module
- First and second strand cDNA synthesis using NEBNext synthesis modules
- Library preparation from 10ng ds-cDNA using an Ovation SP ultralow library system in combination with a Mondrian SP work station
- Library amplification by PCR for 15 cycles
- Sequencing on a 1x50bp program on a Hiseq 2000 or 2500 (Illumina)
- Alignment of files to the mm10 UCSC genome using TopHat splice junction mapper with Bowtie2
- Feature counting and count table production using Rsubread
- Total RNA extraction (not explicitly mentioned)
- Positive control: Not mentioned
- Negative control: Not mentioned
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