Submissions

Submitting RNA

Sample Requirements

General Requirements:

  • Include a DNase treatment with the RNA isolation method - this ensures sample purity and accurate quantitation
  • Samples must be stored in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 7.0-8.0, 0.1 mM EDTA) at -80°C
  • RNA samples must not contain > 1 mM EDTA and must be free of salts and organic contaminants, such as phenol and ethanol since these substances can interfere with library preparation reactions
  • Provide spectrophotometric absorbances from a NanoDrop or similar. A260/A280 and A260/A230 absorbance ratios indicate RNA purity. Values outside these ranges indicate the presence of contaminants
    • A260/A280 ratio of >2.0
    • A260/A230 ratio of 2.0-2.2+

See Evaluating Nucleic Acid Quality for NGS for more information

Illumina Stranded Total RNA Prep with Ribo-Zero Plus, Ligation:

  • 1 - 1000 ng high-quality purified total RNA (RIN ≥ 9) is required in a final volume ≤ 10 μL
    • 10 - 100 ng low-quality RNA (RIN ≥ 2) or FFPE (DV200 > 55%) samples
    • 10 ng minimum for optimal quality & FFPE samples
  • Failure to treat with DNase or inefficient DNase treatment can result in a significant fraction of intergenic reads in the sequence data

Illumina Stranded mRNA Prep, Ligation:

  • 25 - 1000 ng high-quality purified total RNA (RIN ≥ 9) is required in a final volume ≤ 25 μL

Quantification

  • Proper quality control and quantitation requires at least 4 μL of each sample in addition to that required for library preparation
  • Each RNA sample is first evaluated on the Agilent TapeStation 4150 with either RNA or HSRNA ScreenTapes
  • If the samples are of good quality and quantity, the samples will be quantified by Qubit and diluted appropriately for library preparation
  • If the samples are of poor quality or quantity, the customer will be notified with recommendations before moving forward with library preparation

Submitting DNA

Sample Requirements

General Requirements:

  • Samples must be stored in nuclease-free ultrapure water or low TE buffer (10 mM Tris-HCl, pH 8.0-8.5, 0.1 mM EDTA) at RT, +4C, or -20°C
  • DNA samples must not contain > 1 mM EDTA and must be free of salts and organic contaminants, such as phenol and ethanol since these substances can interfere with the tagmentation reaction and result in assay failure
  • Provide spectrophotometric absorbances from a NanoDrop or similar. A260/A280 and A260/A230 absorbance ratios indicate DNA purity. Values outside these ranges indicate the presence of contaminants
    • A260/A280 ratio of 1.8-2.0
    • A260/A230 ratio of 2.0-2.2+

See Evaluating Nucleic Acid Quality for NGS for more information

Illumina DNA Prep, Tagmentation:

  • 1 - 500 ng is required in a final volume ≤ 10 μL
    • 100 - 500 ng minimum for human DNA samples and other large complex genomes
    • 1 ng minimum for small genomes (e.g. microbial)
    • PCR amplicons must be > 150 bp - tagmentation cannot add an adapter directly to the distal end of a fragment; to ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end

Illumina DNA Prep PCR-free, Tagmentation:

  • 25 - 2000 ng high-quality purified DNA is required in a final volume ≤ 25 μL

NEBNext Ultra II FS DNA, with Fragmentation:

  • 0.1 - 500 ng is required in a final volume ≤ 26 μL
  • DNA should be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or nuclease-free water are also acceptable

NEBNext Ultra II DNA, without Fragmentation:

  • 0.5 - 1000 ng is required in a final volume ≤ 50 μL
  • DNA should be sheared in 1X TE and diluted with with 10 mM Tris-HCl, pH 8.0, 1X TE, or 0.1X TE

Quantification

  • Quantification by spectrophotometry is only useful as a ballpark estimate for input into a fluorometric assay

Submitting Individual Libraries

Sample Requirements

  • All libraries to be quantified and sequenced must have Illumina-compatible p5 and p7 sequences and must use the standard NextSeq 1000 read and index primers
  • All libraries to be sequenced in a single run can be pooled if they are of the same preparation type and each library sample has unique and compatible indexes
  • The library prep method and indexes of each library must be provided

Quantification

  • Each library is first evaluated and quantified on the Agilent TapeStation 4150 with either D1000 or HSD1000 ScreenTapes
  • A dilution series is prepared for each library to obtain a working concentration suitable for qPCR
  • Each library is quantified by qPCR on the Roche LightCycler 96 using an Illumina-compatible library quantification kit
  • A 2 nM dilution is then made of each library using Illumina-provided resuspension buffer (RSB) and equal volumes are combined into pools for sequencing

Submitting Pools

Sample Requirements

  • Each library in the pool must satisfy the aforementioned requirements for submitting individual libraries
  • Pools must be at least 2 nM in concentration and a volume greater than 20 μL must be submitted
  • Pools must be prepared with Illumina-provided resuspension buffer (RSB)
  • Each library sample contained in the pool must have unique and Illumina-compatible indexes, preferably unique dual indexes (UDIs)
  • The library prep method (including which commercial kit or whether custom primer sequences were used) and indexes of each library must be provided

Quantification

  • Pools can be quantified by TapeStation and qPCR before loading on the sequencer
  • If the customer declines and provides their own quantification, the Core is not responsible for poor run quality nor resulting differences in sample representation

Submitting Single-Cell/Nucleus Suspensions for 10X

Please see Library Preps - Single Cell for more information about sample preparation and submission requirements.