Sample Submission Guidelines

You are encouraged to set up a meeting to consult for experimental design with the core director, Dr. Xiwei Wu, to determine the optimal platform, library preparation protocol, and sample size. Please fill out the iLab service request form at the time of sample submission. We prefer you dropping off the samples to our core, located at 655 Huntington Drive, but DNA samples can also be dropped off at Beckman basement IGC freezer. Most of the Sanger sequencing users use Beckman freezer for sample drop off. Samples at Beckman freezer are picked up daily. Typical turnaround time for microarray service is 2-3 weeks, and 3-4 weeks for Illumina and PacBio sequencing, 4-6 weeks for single cell sequencing, 1-4 weeks for data analysis. Please note that actual turnaround time might vary depending on the work load and complexity of the project.

 

Please see appropriate sample requirements below if you would like our core staff to prepare the libraries using your samples. Users can also submit prepared libraries to IGC for sequencing. Please consult us to ensure compatibility of the libraries before planning your experiment. Libraries should be minimally quantified by Qubit before submission. IGC will perform Agilent Bioanalyzer analysis to confirm library size distribution if not done by user. Further size selection based purification might be needed with extra charges. Certain libraries (e.g. low complexity, single cell, amplicon, etc.) will be run at low depth on miSeq or iSeq before full depth sequencing when applicable, with additional charges. 

A.  Illumina Short Read Sequencing

DNA Sequencing 

100 ng or more for genome DNA, PCR products, BAC, YAC etc suspended in EB buffer or H2O  in a volume not to exceed 50ul. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2. The DNA amount should be over 250ng for PCR free sequencing library preparation protocol. We will quantify DNA using Qubit fluorometer.

 

Targeted Exome Capture (Agilent Sure Select Protocol)

10ng-200ng (Low Input protocol) or 500ng-2µg (standard protocol) of purified DNA suspended in EB buffer in a volume not to exceed 50ul. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2. We will quantify DNA using Qubit fluorometer. Note that for FFPE samples, higher input (100ng-2ug) is desired. We will apply FFPE DNA restoration kit to repair the DNAs, which improves the pre-capture library yield.

 

Stranded poly(A) mRNA sequencing (mRNA-Seq)
50ng-1ug of Purified total RNA suspended in nuclease-free H2O in a volume less than 50µl. RNA should be as intact as possible. We will verify RNA integrity via an Agilent 2100 Bioanalyzer. RIN ≥8.0 is required for this protocol. 

 

Stranded whole transcriptome sequencing (RNA-Seq) (with RiboErase)
25ng-1ug of purified total RNA (DNA free) suspended in nuclease-free H2O in a volume less than 50µl. RNA should be as intact as possible, but most degraded RNA works fine with this protocol. We will verify RNA integrity via an Agilent Technologies 2100 Bioanalyzer.

 

Stranded FFPE RNA-Seq
Input requirement: 5-50ng of purified total RNA from FFPE samples, or 250pg-10ng of total RNA from other samples. RNA should be suspended in nuclease-free H2O in a volume less than 50µl. DV200 >= 25% is required. We will verify DV200 via an Agilent Technologies 2100 Bioanalyzer. Note that exon content might be lower than better quality RNA, and it requires more read depth to get sufficient coverage.

 

Ultralow input RNA-Seq (unstranded)
For RNA < 250pg, SMART-Seq v4 from Takarabio will be used. This protocol requires intact RNA. RIN>=8 is required.  

 

Small RNA Sequencing
100ng-1ug of purified total RNA suspended in nuclease-free H2O, concentration >20ng/µl. RNA should be isolated using a method that preserves small RNAs. RNA should be as intact as possible, with OD260/280 ratio of 1.8-2 and an OD260/230 ratio over 1.5. 

 

ChIP-seq
>5ng of purified, ChIP-enriched, qPCR-verified DNA suspended in 30ul EB Buffer should be submitted. We will quantify DNA using Qubit fluorometer.

 

ATAC Sequencing
50K cells suspended in DPBS solution with >70% of cell viability. Illumina Nextera protocol is used for the sequencing library preparation. 

 

TCR/BCR sequencing (Immune repertoire sequencing)
*Avoid using heparin for blood sample collection
10ng-3ug of intact total RNA in less than 10µl of nuclease-free H2O, or unfixed 100-10,000 cells in less than 10 µl of DPBS. Takara Bio NucleoSpin RNA XS kit is recommended for RNA extraction from extremely small samples (~100 cells). RIN>8 is desired, but RIN slightly lower will be accepted. 

 

DNA methylation sequencing (BS-seq, RRBS-seq and TAB-seq)
100ng - 2 µg of DNA in less than 25µl of EB buffer, with OD260/280 ratio of 1.8-2 

 

16s rRNA Microbiome sequencing 
*Recommend DNA extraction kit: QIAamp DNA Microbiome kit or QIAamp Fast DNA Stool Mini kit

 

16s rRNA V1-V3, V3-V4, or V4-V5 sequencing: 5-100ng of DNA with OD260/280 ratio of 1.8-2 in less than 10 µl of EB buffer 

 

B.  Single Cell Sequencing With 10x Genomics Chromium

Single cell transcriptome sequencing 

  1. 3’ gene expression with feature barcoding and CRISPR screening
  2. 5’ gene expression with immune profiling (TCR and BCR) and feature barcoding

*Avoid using heparin for blood sample collection.

 

Single cell suspension with cell concentration of 400-1200 cell/µl in DPBS+0.04% BSA (BSA concentration can be up to 0.5%). Cell viability of over 70% is desired.


Samples can be multiplexed with hash-tag antibody from BioLegnd or other companies

 

Sing cell ATAC Sequencing
Single cell suspension with cell concentration of 700-1200 cell/µl in DPBS+0.05% BSA (BSA concentration can up to 0.5%). Cell viability is over 70%.  

If you would like to provide isolated single nuclei, please follow 10xGenomics Nuclei Isolation for Single Cell ATAC Sequencing kits user Guide for nuclei isolation. 

 

Spatial transcriptome
Frozen tissue section on special spatial slides are required. Tissue permeabilizaton condition might need to be optimized. Please contact us for details before initiating a project.

C.  PacBio Long Read Sequencing 

Iso-Seq RNA sequencing
Usually, 500 ng -1 µg of RNA is good enough for QC and sample preparation.  Total and polyA+ RNA must be clean and free from protein and DNA contamination and meet the following minimum requirements: 260/280 ratios must be between 1.9 and 2.1. 260/230 ratios must be above 1.8 (if < 1.8, samples has organic contaminants). We recommend using the QIAGEN RNeasy columns for total RNA purification. 

 

DNA sequencing
Sample input amount depends on library size”

  1. For 20-kb long template using BluePippin Size-Selection System, 10 µg DNA is required.
  2. For 100 - 750 bp template, 1 ug DNA is required.
  3. For 750 bp - 10 kb template, 2 ug DNA is required.
  4. If your sample is very limited (10 - 100 ng), please contact us for special input protocol.
     

The sample concentration above 100 ng/ul is helpful for the 20-kb long template preparation. 260/280 ratios must be between 1.8 and 2.0. 260/230 ratios must be above 2.0 (if < 2, samples has organic contaminants).

 

Genomic DNA can be extracted using any methods that yield high quality DNA. Amplified DNA must be purified, and we recommend using the Qiagen QiaQuick PCR purification protocol with samples eluted in nuclease-free water or the elution buffer provided by in the kit.

 

StrainID rRNA Microbiome Profiling (16s+ITS+23s)
1-3 mg of fresh frozen fecal sample in a screw cap tube is required. If the sample is stored in buffer or is mostly liquid, > 20 ul of sample is required.

D.  Microarray

We provide the genotyping and DNA methylation analysis using Illumina Microarray technologies, and gene expression analysis with Affymetrix’s GeneChip arrays. 

 

Genotyping Analysis  
DNA concentration >50 ng/ul is required. 260/280 ratios must be between 1.8 and 2.0, and 260/230 ratios must be above 2.0.

 

DNA input amount depends on the array format. For Infinium Omni5-4 BeadChip (4 samplers per BeadChip), 500ng purified DNA is required. For smaller chip, such as QC-24 array, 250ng DNA is required. These arrays are compatible with FFPE samples. Samples need to be submitted at multiples of size of subarrays on each chip.

 

Methylation Analysis
Infinium MethylationEPIC BeadChip (8 samples per Bead Chip)
Total > 600ng of purified genomic DNA, with concentration 15 ng/ul is required. 260/280 ratios must be between 1.8 and 2.0, and 260/230 ratios must be above 2.0. Samples need to be submitted at multiples of 8. 

 

Gene Expression Analysis
250pg-10ng purified total RNA (fresh frozen tissue or cells), and 1ng-50ng purified total RNA (FFPE tissue) are required.

E.  Sanger Sequencing 

Log on the LIMS system at dnatools.coh.org

Concentrations:

1. Template – PCR Product:

Input amount depends on the length of the PCR product

  • If your PCR product is < 500bp, calculate the total amount by at least 0.5ng per 100bp, and dilute the sample to 1ng/ul. 
  • If your PCR product is >= 500bp then calculate the total amount by 3ng per 100bp, and dilute the sample in 2ul volume.  

 

For example: If your PCR product size is 600 base pairs, multiply 600 X 0.03 which will give you the total amount of DNA necessary ( 18ng) for sequencing. Then dilute your PCR sample to the correct working concentration of 9ng/ul in 2ul total volume.

 

2. Template – Plasmid

Concentration at 100ng/ul, and 2.0ul is needed per reaction

 

3. Template – BAC Clones
2.5ug/ul concentration for BAC DNA, and 2.0ul is needed per reaction

 

4. Custom Primers
Concentration at 1pmol/ul for PCR or plasmid
10 pmol/ul for BAC sequencing primers
4ul needed per reaction

 

5. Cell Line Validation:
Genomic DNA of 10 ng/ul and >= 2ul. 

Chemistry

  • BigDye_V3.1 – For basic sequencing.  Can read out to approximately 900 base pairs.
  • BigDye_V1.1 – Used only for short (less than 500 base pairs) PCR products when high resolution near the priming site is needed.
  • BigDye_3.0-dGTP – Used only for templates that are likely to form strong secondary structures, hairpin loops, or with high GC content (greater than 75%) or bisulfite-treated samples.  When using this chemistry, please include the Tm of your primer in the comments box.
     

Make sure after submitting the entry form, you print out the table that appears.  The printed form must be placed in the inbox to inform us that the samples are ready to go.  If we do not receive a copy of this form, your samples will not be sequenced.  Please also print another copy for your own reference.


Please label your samples with less than 8 characters if possible.  Write the labels on the cap of the tubes you are submitting. Make sure that the labels on your tubes matches identically to the labels on the order form.


If you need a Chromatograph viewer, you can download one for free from Applied Biosystems.