Materials and methods for preparation of a spatial transcriptomics library
AssigneeIllumina, Inc.
InventorsYue Ding, Lena Storms, Craig April, Yao Xiao, Anustup Poddar, Brittany Flowers, Mats Ekstrand
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Abstract
The present disclosure relates, in general, to materials and methods for improving RNA capture in situ from tissue samples and improved methods for synthesizing cDNA from the captured RNA.
Claims37
A method for isolating RNA from a sample comprising,
- (a) contacting total RNA isolated from the sample with polynucleotide kinase (PNK) to modify 3′ phosphate to a hydroxyl group to generate end repaired total RNA;
- (b) contacting the end repaired total RNA with polyadenylate polymerase (PAP) and adenosine nucleotides to generate polyadenylated total RNA;
- (c) capturing the polyadenylated total RNA on a substrate comprising one or more oligonucleotides comprising poly T sequences;
- and
- (d) eluting the polyadenylated total RNA from the substrate.
A method for preparing an RNA library from a tissue sample comprising,
- (a) contacting total RNA isolated from the sample with polynucleotide kinase (PNK) to modify 3′ phosphate to a hydroxyl group to generate end repaired total RNA;
- (b) contacting the end repaired total RNA with polyadenylate polymerase (PAP) and adenosine nucleotides to generate polyadenylated total RNA;
- (c) releasing the polyadenylated total RNA from the tissue sample;
- (d) capturing the polyadenylated total RNA on a substrate comprising one or more oligonucleotides comprising a poly T sequence;
- and
- (e) generating an RNA library from the polyadenylated total RNA using a RNA library prep kit.
A method for preparing an mRNA transcriptome library from a tissue sample comprising,
- (a) contacting total RNA isolated from the sample with polynucleotide kinase (PNK) to modify 3′ phosphate to a hydroxyl group to generate end repaired total RNA;
- (b) contacting the end repaired total RNA with polyadenylate polymerase (PAP) and adenosine nucleotides to generate polyadenylated total RNA;
- (c) releasing the polyadenylated total RNA from the tissue sample;
- (d) capturing the polyadenylated total RNA on a substrate comprising one or more oligonucleotides comprising a poly T sequence;
- (e) depleting ribosomal RNA from the total RNA to leave polyadenylated mRNA;
- and
- (f) generating an mRNA library from the polyadenylated mRNA using a mRNA library prep kit.
The method of any one of the preceding claims, wherein the substrate is a bead, a bead array, a spotted array, a flow cell, clustered particles arranged on a surface of a chip, a film, and a plate.
The method of any one of the preceding claims, wherein the sample is a fresh frozen tissue sample or a formalin-fixed paraffin embedded (FFPE) sample.
The method of any one of the preceding claims, wherein the polyA tail is between 3 and 50 nucleotides.
A method for improving capture efficiency of mRNA transcripts for in situ mRNA transcript library preparation comprising,
- (a) capturing mRNA transcripts from a sample on a substrate;
- (b) contacting the substrate with a high processivity reverse transcriptase (RT) to generate a first cDNA strand complementary to the mRNA transcripts;
- (c) contacting the first cDNA strand with a DNA polymerase to generate a second cDNA strand complementary to the first cDNA strand;
- and
- (d) amplifying the second strand cDNA to form a PCR template and isolating the PCR template.
A method for improving capture efficiency of mRNA transcripts for in situ mRNA transcript library preparation comprising,
- (a) capturing mRNA transcripts from a sample on a substrate;
- (b) contacting the substrate with a reverse transcriptase (RT) to generate a first cDNA strand complementary to the mRNA transcripts;
- (c) contacting the first cDNA strand with a high processivity reverse transcriptase (RT) or high processivity DNA polymerase to generate a second cDNA strand complementary to the first cDNA strand;
- and
- (d) amplifying the second strand cDNA to form a PCR template and isolating the PCR template.
A method for improving capture efficiency of mRNA transcripts for in situ mRNA transcript library preparation comprising,
- (a) capturing mRNA transcripts from a sample on a substrate;
- (b) contacting the substrate with a high processivity reverse transcriptase (RT) to generate a first cDNA strand complementary to the mRNA transcripts;
- (c) contacting the first cDNA strand with the high processivity reverse transcriptase (RT) or high processivity DNA polymerase to generate a second cDNA strand complementary to the first cDNA strand;
- and
- (d) amplifying the second strand cDNA to form a PCR template and isolating the PCR template.
A method for improving the nucleotide length of polynucleotides used in generating an in situ transcriptome library comprising,
- (a) capturing mRNA transcripts from a sample on a substrate;
- (b) contacting the substrate with a high processivity reverse transcriptase (RT) to generate a first cDNA strand complementary to the mRNA transcripts;
- (c) contacting the first cDNA strand with a high processivity reverse transcriptase (RT) or high processivity DNA polymerase to generate a second cDNA strand complementary to the first cDNA strand;
- (d) amplifying the second strand cDNA to form a PCR template and isolating the PCR template.
A method for preparing an mRNA transcriptome library from a tissue sample comprising,
- (a) contacting total RNA isolated from the sample with polynucleotide kinase (PNK) to modify 3′ phosphate to a hydroxyl group to generate end repaired total RNA;
- (b) contacting the total RNA with polynucleotide kinase (PNK) to modify a 3′ phosphate to a hydroxyl group to generate end repaired total RNA;
- (c) contacting the end repaired total RNA with polyadenylate polymerase (PAP) and adenosine nucleotides to generate polyadenylated total RNA;
- (d) releasing the polyadenylated total RNA from the tissue sample;
- (e) capturing the polyadenylated total RNA on a substrate comprising one or more oligonucleotides comprising a poly T sequence;
- (f) depleting ribosomal RNA from the total RNA leaving polyadenylated mRNA;
- (g) contacting the polyadenylated mRNA with a reverse transcriptase (RT) to generate a first cDNA strand complementary to the mRNA transcripts;
- (h) contacting the first cDNA strand with a high processivity reverse transcriptase (RT) or high processivity DNA polymerase to generate a second cDNA strand complementary to the first cDNA strand to generate PCR templates;
- (i) eluting the PCR templates;
- and
- (j) generating an mRNA library from the PCR templates.
Dependencies are inferred from the claim text.