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NGS Sample Preparation

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The following sample preparation guidelines apply to our NGS assays:

Application: Sequencing only
Sample Type: User-prepared library
Recommended Amount: > 10 nM, > 30 ul (individual libraries); > 10nM, > 70 ul (pool)
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2

 

Application: Total RNA-seq
Sample Type: DNase-treated, total RNA
Recommended Amount: > 25 ng , < 9 ul
Minimum Amount: 1 ng good-quality RNA; 100 ng degraded RNA; < 9 ul;
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.2, A260/230: 1.45 - 2.2
Quality Requirements: RIN > 7.5

Application: PolyA RNA-seq
Sample Type: DNase-treated, total RNA
Recommended Amount: > 600 ng, < 45 ul
Minimum Amount: 100 ng, < 45 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.2, A260/230: 1.45 - 2.2
Quality Requirements: RIN > 7.5

Application: miRNA-seq
Sample Type: DNase-treated, total RNA. Ensure retention of small RNAs during RNA isolation.
Recommended Amount: 250 ng, < 5ul
Minimum Amount: 10 ng, < 5 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.2, A260/230: 1.45 - 2.2
Quality Requirements: RIN > 7.5

Application: Low Input RNA-seq
Sample Type: DNase-treated, total RNA
Recommended Amount: > 1 ng, < 5 ul
Minimum Amount: 500 pg, < 5 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.2, A260/230: 1.45 - 2.2
Quality Requirements: RIN > 7.5

Application: ChIP-seq
Sample Type: ChIP DNA, Input DNA
Recommended Amount: > 5 ng, < 10 ul
Minimum Amount: 50 pg, < 10 ul
Buffer: EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: Input DNA size range: 100 – 1000 bp.

Application: Whole Genome Bisulfite Sequencing
Sample Type: genomic DNA
Recommended Amount: > 500 ng, < 15 ul
Minimum Amount: 200 ng, < 15 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: DIN > 7.5

Application: Whole Exome Capture
Sample Type: genomic DNA
Recommended Amount: > 5 ug, < 50 ul
Minimum Amount: 200 ng, < 50 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: DIN > 7.5

Application: Whole Genome Sequencing
Sample Type: genomic DNA
Recommended Amount: > 150 ng, < 10 ul
Minimum Amount: 50 pg, < 10 ul
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: DIN > 7.5

Application: Whole Genome Sequencing (PCR free)
Sample Type: genomic DNA
Recommended Amount: > 2.5 ug, < 30 ul
Minimum Amount: 50 ng, < 30 ul
Buffer: H2O
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: DIN > 7.5

Application: Amplicon-seq
Sample Type: PCR product
Recommended Amount: > 2.5 ng, < 5 ul
Minimum Amount: 1 ng
Buffer: H2O, EB, low TE (< 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: > 300 bp