Truseq stranded mrna seq

Mouse embryonic stem cells were collected with accutase (5 min, 37 °C) and counted. Cells (3 × 10⁵) were lysed with 300 µl TRIzol reagent. RNA was extracted using the Direct-Zol RNA extraction kit from Zymo. Library preparation was performed after Illumina TruSeq Stranded mRNA-seq according to the manufacturer protocol. Reads were mapped to the Mus musculus genome (build mm9) using STAR^{58 (link)}, using the following options: --outSJfilterReads Unique --outFilterType BySJout --outFilterMultimapNmax 10 --alignSJoverhangMin 6 --alignSJDBoverhangMin 2 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate --seedSearchStartLmax 50 --twopassMode basic. Gene expression was quantified using qCount from QuasR package^{59 (link)} using the ‘TxDb.Mmusculus.UCSC.mm9.knownGene’ database for gene annotation (Bioconductor package: Carlson M and Maintainer BP. TxDb.Mmusculus.UCSC.mm9.knownGene: Annotation package for TxDb object(s); R package v.3.2.2). Active promoters were defined as genes with log₂[RPKM + 0.1] higher than 1.5.

RNA quantity was assessed using picogreen. Samples were then diluted to an equal concentration in 96-well plates. RNA was then used for gene expression profiling using the bulk RNA barcoding and sequencing (BRB-seq) approach recently developed by our lab [65 ]. This protocol is able to provide high-quality 3′ transcriptomic data by implementing an early multiplexing scheme as in single-cell protocols and at a fraction of the cost of its competitors (e.g., 10-fold lower than Illumina Truseq Stranded mRNA-seq). In short, the BRB-seq protocol starts with oligo-dT barcoding, without TSO for the first-strand synthesis (reverse transcription), performed on each sample separately. Then all samples are pooled together after which the second-strand is synthesized using DNA PolII Nick translation. The sequencing library is then prepared using cDNA tagmented by an in-house produced Tn5 transposase preloaded with the same adapters (Tn5-B/B) and further enriched by limited-cycle PCR with Illumina compatible adapters. Libraries are then size-selected (200–1000 bp), profiled using a High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474), and measured using a Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851). Finally, 6–8 pg of libraries was sequenced twice with Illumina NextSeq 500 with 21 cycles for read 1 (R1) and 101 cycles for read 2 (R2), only for the second sequencing.

The BRB-seq is a technique for multiplexed RNA-seq [25 (link)] which is able to provide high-quality 3′ transcriptomic data at a low cost (e.g., 10-fold lower than Illumina Truseq Stranded mRNA-seq). The data (fastq files) generated from BRB-seq are multiplexed and asymmetrical paired reads. Read R1 contains a 6-bp sample barcode, while read R2 contains the fragment sequence to align to the reference genome.