Single cell gene expression studies have increased our
understanding of the function of individual cells in normal
development and disease. Due to the limited messenger
ribonucleic acid (mRNA) amount in a cell, ultrasensitive
methods are necessary for reliable detection. Single cell RNA
sequencing (RNA-seq) has been applied for profiling entire
transcriptomes.1−7 Many methods use Moloney murine
leukemia virus (MMLV) template switching (TS) to
incorporate a universal primer site during oligo-dT primed
complementary DNA (cDNA) synthesis when reverse transcription
(RT) reaches mRNA 5′ ends.8 Universal polymerase
chain reaction (PCR) is subsequently applied to amplify
cDNAs for sequencing. TS has become increasingly popular
because of fewer protocol steps, high overall efficiency, and the
ability to represent full-length transcripts. However, questions
regarding the sensitivity, accuracy, and technical reproducibility
remain.
Single cell gene expression studies have increased ourunderstanding of the function of individual cells in normaldevelopment and disease. Due to the limited messengerribonucleic acid (mRNA) amount in a cell, ultrasensitivemethods are necessary for reliable detection. Single cell RNAsequencing (RNA-seq) has been applied for profiling entiretranscriptomes.1−7 Many methods use Moloney murineleukemia virus (MMLV) template switching (TS) toincorporate a universal primer site during oligo-dT primedcomplementary DNA (cDNA) synthesis when reverse transcription(RT) reaches mRNA 5′ ends.8 Universal polymerasechain reaction (PCR) is subsequently applied to amplifycDNAs for sequencing. TS has become increasingly popularbecause of fewer protocol steps, high overall efficiency, and theability to represent full-length transcripts. However, questionsregarding the sensitivity, accuracy, and technical reproducibilityremain.
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