METHODS AND COMPOSITIONS FOR cDNA SYNTHESIS AND SINGLE-CELL TRANSCRIPTOME PROFILING USING TEMPLATE SWITCHING REACTION

摘要

This application discloses methods for cDN′A synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (INA) at the TSO 3′ end, using a methyl group donor, and/or a MgCb concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3′-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.

主权项

1. A method for preparing DNA that is complementary to an RNA molecule, comprising the steps of:
annealing a cDNA synthesis primer to said RNA molecule and synthesizing a first cDNA strand to form an RNA-cDNA intermediate; conducting a reverse transcriptase reaction by contacting said RNA-cDNA intermediate with a template switching oligonucleotide (TSO), wherein the TSO comprises a locked nucleic acid (LNA) at its 3′-end, under conditions suitable for extension of the first DNA strand that is complementary to the RNA molecule, rendering it additionally complementary to the TSO; and optionally amplifying said DNA strand that is complementary to said RNA molecule and said template switching oligonucleotide using an oligonucleotide primer, wherein optionally said RNA is total RNA in a cell.