V4 chemistry

llumina TruSeq mRNA libraries were generated and sequenced with 50–65 base single reads on a HiSeq 2500 using V4 chemistry (Illumina Inc., San Diego)^{50 (link)}. The resulting reads were trimmed using Cutadapt (version 1.15) to remove any remaining adapter sequences and to filter reads shorter than 20 bp after trimming to ensure good mappability. The trimmed reads were aligned to the GRCm38 reference genome using STAR (version 2.5.3a). QC statistics from Fastqc (version 0.11.5) and the above-mentioned tools were collected and summarized using Multiqc (version 1.1). Gene expression counts were generated by featureCounts (version 1.5.2) using gene definitions from Ensembl GRCm38 version 89. Normalized expression values were obtained by correcting for differences in sequencing depth between samples using DESeqs median-of-ratios approach and then log-transforming the normalized counts.

Total RNA was extracted using TRIzol reagent (Ambion life technologies) according to the manufacturer’s instructions. Quality and quantity of total RNA was assessed on a 2100 Bioanalyzer using a Nano chip (Agilent). Total RNA samples having RIN values >8 were subjected to library generation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc.) according to the manufacturer's instructions. Libraries were sequenced on a HiSeq2500 using V4 chemistry (Illumina Inc.), and reads (65bp single-end), were aligned against the human reference genome (hg38) using TopHat (version 2.1.0), allowing the spanning of exon-exon splice junctions. TopHat was supplied with a known set of gene models (Ensembl version 77). Samples were generated using a stranded library preparation protocol, in which TopHat was guided to use the first-strand as the library-type. Tophat was run with bowtie 1 version 1.0 and the additional parameters “—prefilter-multihits” and “—no coverage”. In order to count the number of reads per gene, a custom script (ItreeCount) was used. This script is based on the same concept as HTSeq-count and has comparable output. ItreeCount generates a list of the total number of uniquely mapped sequencing reads for each gene that is present in the provided Gene Transfer Format (GTF) file.

116 ng of total RNA was depleted using the Illumina Ribo-zero rRNA Removal Kit (Bacteria) and purified with Ampure XP beads. Successful depletion was confirmed using Qubit and Agilent 2100 Bioanalyzer. All of the depleted RNA was used as input material for the ScriptSeq v2 RNA-Seq Library Preparation protocol. Following 15 cycles of amplification the libraries were purified using Ampure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyzer.
The final libraries were pooled in equimolar amounts using the Qubit and Bioanalyzer data. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa on a Roche Light Cycler LC480II according to manufacturer's instructions. Sequencing was performed at the Centre for Genomic Research, University of Liverpool, on one lane of the Illumina HiSeq 2500 2x125 bp using v4 chemistry (Illumina).