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AssigneeTakara Bio USA, Inc.
InventorsCynthia Chang, Magnolia Bostick
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Methods of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample are provided. Aspects of the methods include combining the RNA sample with a first strand cDNA primer and a template switch oligonucleotide under first strand cDNA synthesis conditions, where one of the first strand cDNA primer and the template switch oligonucleotide includes a first post-tagmentation amplification primer binding domain. The resultant product is subjected to amplification conditions sufficient to produce a double stranded cDNA, which is then tagmented with a transposome that includes a second post-tagmentation amplification primer binding domain. The tagmented sample is then subjected to amplification conditions using first and second post-tagmentation amplification primers that include sequencing platform adapter constructs to produce a NGS library. Aspects of the invention further include compositions produced by the methods and kits that find use in practicing the methods.
A method of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample, the method comprising:
The method according to any of the preceding claims, wherein the first and second pre-tagmentation amplification primer binding domains are identical and the first and second pre-tagmentation amplification primers are identical.
The method according to any of the preceding claims, wherein the transposase comprises a Tn5 transposase.
The method according to any of the preceding claims, wherein the method further comprises pooling the double stranded product cDNA with a second double stranded product cDNA to produce a pooled cDNA sample, and then tagmenting the pooled cDNA sample.
The method according to any of the preceding claims, wherein the RNA sample is one that is produced from a single cell.
The method according to any of the preceding claims, wherein the method further comprises subjecting the NGS library to an NGS protocol.
The method according to any of the preceding claims, wherein the method further comprises quantitating one or more RNA species of the RNA sample.
A composition comprising a template mRNA and a template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein one of the first strand cDNA comprises and template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain.
A kit comprising:
Dependencies are inferred from the claim text.