A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design

Abstract

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

Single-cell transcriptome analysis of Physcomitrella leaf cells during reprogramming using microcapillary manipulation

“…In this study, we employed the latter technique for the 1cell-DGE based on our results (Additional file 2: Supplementary Figure S2). While we found template switching to be less effective than poly(dA) tailing, this could be improved by the use of a short template-switching oligo and locked nucleic acid (LNA)-linked nucleotides [46-48], and might be suitable for use with 1cell-DGE following such improvements.…”

FB5P-seq: FACS-based 5-prime end single-cell RNAseq for integrative analysis of transcriptome and antigen receptor repertoire in B and T cells

“…Although this may be low for some genes in some cell types, in our analyses on lymphocytes, only the CD74 gene showed recurrent saturating levels in Mem B cells and GC B cells (not shown). Longer UMI sequences could be used in the TSO design to prevent saturation issues 10,24 , but template switching efficiency may be affected by increased TSO length 35 . In our analyses of human lymphocytes, we have used very low levels of ERCC spike-ins (0.025 pg per well, corresponding to a 1:2,000,000 final dilution) compared to other scRNA-seq protocols 11,12,36 .…”

“…We based the design of the FB5P-seq experimental workflow on existing full-length 8 and 5'-end 9,24 scRNA-seq protocols. The main originalities in FB5P-seq were to perform cellspecific barcoding and incorporate 5 bp UMI during reverse transcription, and sequence the 5'-ends of amplified cDNAs from their 3'-end, and not from the transcription start site ( Figure 1A-B).…”

Decode-seq: a practical approach to improve differential gene expression analysis

“…BRB-seq libraries contain a poly(T) stretch introduced by the reverse transcription primer. Sequencing through such a low diversity region typically yields poor read qualities [19]. There are three ways to deal with this problem.…”