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NGS (GARP Core)

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NGS Sample Preparation

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Please add 5-10 ul extra volume to the requirements below for RNA/DNA samples to be submitted for Sequencing Library Prep. GARP requires a single tube to be used for the Quality Control test and Library prep.

The following sample preparation guidelines apply to our NGS assays. If your samples don’t meet the requirements, please schedule a meeting with the Lab Manager or Core Director – we have solutions for challenging sample types (e.g. low input, degraded).

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: > 500 ng @ 25-50 ng/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: > 500 ng @ 10-50 ng/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: 3’ RNA-seq
Sample Type: Total RNA
Recommended Amount: > 500 ng @ 25 – 100 ng/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 @ 25 – 50 ng/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: > 10 ng @ > 1 ng/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: > 1000 ng @ > 50 - 100 ng/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: > 200 ng @ 10 – 50 ng/ul
Buffer: H2O, EB
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: 1.45 - 2.2
Quality Requirements: DIN > 7.5

Application: 10X Visium
Sample Type: Fresh-frozen tissue. The recommended freezing method uses an isopentane and liquid nitrogen bath.
Quality Requirements: RIN > 7

To determine the coverage needed for prepared 10X Genomics Single Cell Libraries, use these instructions.

Upon completion of NGS orders, users must download data immediately upon availability. Raw data files will be deleted from our storage three months following data release.