SMART-Seq mRNA Long Read

Description

SMART-Seq mRNA Long Read generates full-length, barcoded cDNA directly from 11,000 intact cells or 10 pg100 ng of total RNA in a convenient input volume of 19.5 mul. The full workflow can be completed within 2 days. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back Library preparation workflow for the SMART-Seq mRNA Long Read kit.Starting with high-integrity RNA RIN8 or intact cells, first-strand cDNA synthesis is primed by the SMART-Seq LR Oligo-dT and performed by an MMLV-derived reverse transcriptase RT . Upon reaching the 5 end of each mRNA molecule, the RT adds nontemplated nucleotides to the first-strand cDNA. The SMART-Seq LR TSO contains a sequence that is complementary to the nontemplated nucleotides added by the RT. This primer hybridizes to the first-strand cDNA. In the template switching step, the RT uses the remainder of the SMART-Seq LR TSO as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. The first-strand cDNA is then barcoded by forward and reverse primers from the SMART-Seq Long Read Index Kit 96 rxns and amplified by the first PCR; a second PCR enriches for barcoded fragments. Samples are pooled and end-prepped, and sequencing adapters are ligated using the Ligation Sequencing Kit V14 ONT . After sequencing, samples are basecalled and demultiplexed using ONTs Guppy. Downstream data analysis was performed using Cutadapt, Minimap2, SAMtools, Bedtools, and Salmon. Back SMART-Seq mRNA LR generates barcoded cDNA with a wide range of fragment lengths.SMART-Seq mRNA LR workflow was used to create cDNA from 10 pg or 10 ng total mouse brain RNA n = 8 . cDNA size distribution was measured on a 2100 Bioanalyzer Agilent Technologies using an Agilent High Sensitivity DNA Kit.Panel A. Barcoded cDNA was pooled per input, and libraries were generated using the Ligation Sequencing Kit V14. Libraries were sequenced on a MinION Flow Cell ONT for 72 hr. Samples were basecalled and demultiplexed using ONTs Guppy and read-length distribution was plotted using MS-Excel.Panels B & C. Read-length distributions are shown for a representative sample of 10 pg Panel B and 10 ng Panel C total mouse brain RNA. Back SMART-Seq mRNA Long Read demonstrates high sensitivity across a broad range of inputs.To evaluate the performance, cDNA and sequencing libraries were generated from 10 pg and 10 ng total mouse brain RNA MBR using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using ONTs Guppy, and reads per barcode were downsampled to 400,000 reads. The read distribution for 10 pg and 10 ng MBR input.Panel A.is similar, showing consistent performance across the input range n = 8 for 10 ng; n = 7 for 10 pg .Panel B.Reads of the 10 pg MBR dataset downsampled to assess gene and transcript sensitivity.Panel C.10 ng MBR dataset downsampled to assess gene and transcript sensitivity. While neither of the two conditions 10 pg and 10 ng were sequenced until saturation, both highlight the high gene and transcript sensitivity across read depths. Back SMART-Seq mRNA LR is highly reproducible across replicates for a broad range of inputs.The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates. Barcoded cDNA of all 96 samples were pooled, and libraries were generated and sequenced according to the workflow. Back SMART-Seq mRNA LR yields even gene-body coverage for ultra-low inputs.10 pg and 10 ng MBR samples underwent the SMART-Seq mRNA LR workflow. Gene-body coverage was assessed for the average of eight replicates of 10 pg and 10 ng MBR samples. Back SMART-Seq mRNA LR is compatible with direct cell inputs.cDNA was generated from single K562 cells or 1,000 K562 cells using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using Guppy, and reads per barcode were downsampled to 300,000 reads.Panel A.The average gene count for each condition is shown above the corresponding bar n = 8 for single cell and n = 2 for 1,000 cells .Panel B. Pearson correlation was calculated from the gene matrix across eight single K562 cells.Panel C.Downsampling analysis of single K562 cells shows gene and transcript count per read depth. Back SMART-Seq mRNA LR detects full-length isoforms and gene fusions.cDNA was generated using the SMART-Seq mRNA LR workflow. Basecalling and demultiplexing were performed using ONTs G uppy, and reads were aligned using Minimap2.Panels A & B.Isoforms ofSnap25 Panel A andNbr1 Panel B detected from 10 pg MBR input are visualized in IGV.Panel C.NUP214::XKR3gene fusions detected from single-cell input are visualized in IGV. Back SMART-Seq mRNA LR is compatible with automation and miniaturization.The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates manually on the benchtop with the full reaction volumes or automated at 1/8-volume on a mosquito HV SPT Labtech . Barcoded cDNA of all 96 samples per experiment was pooled, and libraries were generated and sequenced for 72 hr according to the workflow. Samples were basecalled and demultiplexed using ONT Guppy and aligned with Minimap2.Panel A.Render of the mosquito HV liquid handler.Panel B.Gene counts were assessed with Salmon.Panel C. Mapping statistics of manually prepared cDNA compared to cDNA prepared using the mosquito HV. 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