NGS Sample Preparation
To determine the coverage needed for prepared 10X Genomics Single Cell Libraries, please 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.
Application: Sequencing only
Sample Type: User-prepared library
Recommended Amount: > 10 nM, > 30 µl (individual libraries); > 10nM, > 70 µl (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 with ribodepletion
Sample Type: DNase-treated, total RNA
Recommended Amount: > 500 ng @ 25-50 ng/µl
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/µl
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/µl
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/µl
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/µl
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/µl
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: 10X Visium
Sample Type: Fresh-frozen tissue. The recommended freezing method uses an isopentane and liquid nitrogen bath; please consult the 10X Tissue Preparation Guide. FFPE.
Recommended Amount: 2 cores (5mm length) or 2 x 10 µm scrolls for RNA extraction & QC. Provide tissue blocks or 5 μm sections (total 5 mm2) on a glass slide (Epredia Shandon Colorfrost Plus Slides – PN 6776214, Fisher SuperFrost Slides - PN 12-544-7, Sigma-Aldrich Poly-Prep Slides - PN P0425-72EA, VWR SuperFrost Plus Slides - PN 48311-703) for the GEX assay.
Buffer: N/A
Quality Requirements: RIN > 7 (FF), %DV200 > 30% (FFPE).
Application: Nanopore cDNA-seq
Sample Type: DNase-treated, total RNA or polyA-enriched
Recommended Amount: 200 ng (total) or 4 ng (polyA-enriched) in < 20 µl
Buffer: Elution Buffer, 10 mM Tris-Cl, pH 8.5
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: > 2.0
Quality Requirements: RIN > 7.5 (when total RNA is provided)
Application: Nanopore Direct RNA-seq
Sample Type: DNase-treated, total RNA
Recommended Amount: >3 ug in < 30 µl
Buffer: Elution Buffer, 10 mM Tris-Cl, pH 8.5
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: > 2.0
Quality Requirements: RIN > 7.5
Application: Nanopore Whole Genome Seq
Sample Type: gDNA
Recommended Amount: 3-5 ug in < 50 µl
Buffer: Nuclease-free water, Low TE
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: > 2.0
Quality Requirements: DIN > 7.5
Application: Poly(A) enrichment
Sample Type: total RNA
Recommended Amount: 2.5µg - 5 µg in 10µl
Buffer: Elution Buffer, 10 mM Tris-Cl, pH 8.5
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: > 2.0
Quality Requirements: RIN > 7.5
Application: Multiplexed RNA modification sequencing
Sample Type: total RNA or poly (A) enriched, DNase-treated
Recommended Amount: 4- 5 µg of total RNA in < 20 µl or >20ng poly (A) in <20µl
Or 1ug of total RNA <30ul if prepped with Total RNA workflow
Buffer: Low TE (10nM Tris pH 7.0, 0.1mM EDTA)
Purity Requirements: A260/280: 1.75 - 2.0, A260/230: > 2.0
Quality Requirements: RIN > 7.5 (NA for PolyA enriched)