5/7/2023 0 Comments Step seq 2![]() In this analysis on 100 pg of RNA, the number of detected genes also increased, though not significantly (Additional file 1: Figure S1a), which likely reflects that most of the additionally identified transcripts are of genes already detected using the longer primer. Use of the shortened primers indeed improved the sensitivity to 10.6 %, detecting more transcripts (Additional file 1: Figure S1). This was done by reducing the length of the barcode from eight to six nucleotides, as well as shortening the T7 promoter and the Illumina 5’ adaptor. We show that CEL-Seq2 works well on different platforms, and compare it to previously published methods.įirst, we sought to increase the efficiency of the reverse transcription (RT) reaction by shortening the CEL-Seq primer from 92 to 82 nucleotides, despite the addition of six UMI nucleotides. Here we introduce CEL-Seq2, which has been optimized for higher sensitivity, less hands-on time, and lower price. We use early barcoding, enabling highly-multiplexed analysis, and 3′ end tagging enabling accurate estimation of expression levels without having to account for gene length and with fewer sequencing reads required. The CEL-Seq method is the first method to use in vitro transcription (IVT) for the amplification, thereby eliminating the requirement for a template-switch step which is thought to reduce efficiency. Each sample is processed individually, and the reaction was optimized for full transcript sequencing. Smart-Seq used the same template switching mechanism as STRT, but without the early barcoding. The STRT method introduced early barcoding at the reverse transcription stage, thereby enabling highly-multiplexed analyses, and adapted a template switching mechanism based on the ability of the reverse transcriptase to tag the end of the cDNA, eliminating the need for the polyadenylation reaction. Each sample is individually converted to a library for sequencing. After polyadenylation of the resulting cDNA, a second polyT primer with a different anchor is used to obtain double stranded DNA, which is then PCR-amplified. , using a polyT primer with an anchor sequence to select for the cell’s mRNA. Single-cell RNA-Seq was first introduced by Tang et al. Single-cell transcriptomics is a transformative method with tremendous potential to illuminate the complexities of gene regulation.
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