Nucleic Acid Isolation
Nucleic Acid extractions are performed on our QIASymphony instrument.
Blood Samples for RNA extractions
Total RNA will be extracted using the QIAsymphony PAXgene Blood RNA Kit (https://www.qiagen.com/us/shop/sample-technologies/rna/mirna/qiasymphony-paxgene-blood-rna-kit/#orderinginformation). All blood samples (2.5ml) should be collected in PAXgene Blood RNA Tubes (https://www.preanalytix.com/products/blood/RNA/paxgene-blood-rna-tube). Immediately after blood collection, gently invert the PAXgene Blood RNA Tubes 8–10 times. Store the PAXgene Blood RNA Tubes upright at room temperature (18−25 ̊C) for a minimum of 2 hours and a maximum of 72 hours before transferring to freezer. The PAXgene Blood RNA Tubes can be stored at −20 ̊C and below. If tubes are to be kept at temperatures below −20 ̊C, freeze them first at −20 ̊C for 24 hours, then transfer them to −80 ̊C.
Tubes should be delivered to us frozen. Note that the frozen PAXgene Blood RNA Tubes are very fragile and break easily.
Tubes containing material for isolation must be clearly labeled with your DUGSIM order number and sample number (“4596_S001”). For human blood samples, please make sure to provide us with your IRB number to confirm that your project has the appropriate IRB approval.
Cell pellets for RNA extractions
Total RNA from cell pellets will be extracted using the Qiagen QIAsymphony RNA Kit (https://www.qiagen.com/us/shop/automated-solutions/qiasymphony-rna-kit/#orderinginformation). Cell pellets should have between 1-3 million cells.
Cells should be pelleted in Sarstedt 2.0 mL tubes - cat # 72.693 or 72.608
sequencing library preparation
Samples may be suspended in different types of buffer, including 10mM Tris, dH2O, or Qiagen EB. We do however prefer when samples are suspended in dH2O. It will allow us to concentrate the samples when necessary without having to worry about concentrating the salts present in other types of buffer. High salt (EDTA) concentration can interfere with the various enzymes that are used during library preparation, especially for PacBio sequencing.
Plates and Tubes:
When submitting one sample, please submit in a 1.5mL Eppendorf tube. Make sure to write the sample number listed on your order sample sheet on the top the tube.
Should you submit more than one sample, please use polypropylene PCR plates. Plates should be labeled with the order number and sample number range clearly marked (e.g 4596_S01-S96), and should be sealed firmly with a foil plate seal capable of withstanding -80 C temperatures. We recommend the Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626). If you have more than one plate please also number the plates (e.g 4596_S01-S96_Plate1, 4596_S97-S192_plate2). For the plates, we recommend the Olympus 96-Well PCR Plate, Semi-Skirted (Genesee Scientific # 27-108).
Any plate submitted must be clear and semi-skirted! We will no accept full-skirted, deep well, round-bottom, or flat-bottom plates!
Samples should be loaded on the plate in columns and not in rows! Sample 1 should be in well A1, samples 2 in well B1, samples 3 in C1, etc.
Sample Concentration and Volume:
All samples undergo quality check (QC) before going into library preparation. Make sure to include enough volume to allow for 5 uL to be used in QC. The inputs listed below should remain after the completion of this QC.
|Library Type||Lib prep kit used||Input Type||Input amount recommended by manufacturer||minimum volume||maximum volume||recommended amount|
|DNA-Seq||Kapa Hyper Prep||genomic DNA||1 ng - 1 ug||20 µl||50 µl||≥ 500 ng|
|Stranded mRNA-Seq||Kapa Stranded mRNA-seq kit||total RNA||100 ng - 4 ug RIN ≥ 7||20 µl||50 µl||≥ 500 ng|
|Stranded Total RNA-Seq with Ribo-zero||Illumina Tru-Seq Stranded Total RNA-seq kit W/ Ribo-Zero||total RNA||100 ng - 1 ug RIN <7 is acceptable||10 µl||10 µl||≥ 500 ng|
|Low RNA input RNA-Seq - not stranded||Clontech SMARTer Ultra-low input mRNA-seq kit followd by Kapa Hyper Prep||total RNA||10pg - 10ng RIN ≥ 7||9.5||9.5||≥ 10 ng|
|smRNA-Seq||QiaSeq miRNA Library Prep||total RNA||1 ng - 10ng RIN ≥ 7||5ul||5 µl|
|Mate-Pair||Nextera Mate-Pair Sample Prep||genomic DNA||4 ug||20 µl||48 µl||≥ 4 ug|
|blood mRNA-seq||Nugen Universal Plus mRNA-seq||Total RNA from blood||10 ng - 1 ug RIN ≥ 7||10 µl||50 µl||≥ 500|
|ChIP-Seq||Kapa Hyper Prep||ChIP enriched DNA||1 ng - 1 ug||10 µl||50 µl|
We do accept prepared libraries for sequencing. Prepared libraries should be sent at ≥10 nM. A volume of 15ul at the minimum is required. Concentration should ideally be determined using fluorometric means (picogreen/Qubit).
|Library type||Lib Prep Kit we will use||Input Type||Input amount recommended by manufacturer||minimum volume||maximum volume|
|15-20kb insert library||SMRTbell Express Template Prep Kit||gDNA||≥ 10 ug||50 µl||150 µl|
|Amplicons||SMRTbell Template Prep Kit 1.0||PCR amplicons||1 ug - 2 ug||50 µl||150 µl|
|IsoSeq||Clontech SMARTer PCR cDNA Syntheis kit followed by SMRTbell Template Prep Kit 1.0||Total RNA||600 ng - 1ug RIN ≥ 7||10ul||10 ul|
We do not accept prepared libraries for PacBio sequencing.
The PacBio template preparation process does not utilize amplification techniques. Therefore, the input DNA quality will be directly reflected in the sequencing results. Any DNA damage (e.g., basic sites, nicks, interstrand crosslinks) or contaminants (e.g., single-stranded DNA, RNA, proteins, dyes, or salts, phenol) present in the input material will impair performance of the system. PacBio recommends Qiagen MagAttract HMW gDNA extraction kits. gDNA samples need an OD 260/280 ratio of approximatively 1.8 to 2.
For PCR products, gel cuts may be done, but the gel can not be exposed to ethidium bromide and UV light. Use instead CYBR green and blue light. PCR products must be cleaned prior to submission using either AMPure XP beads or Qiagen PCR cleanup spin columns.
Ensure that your DNA sample:
- Is double-stranded. Single-stranded DNA will not be made into a SMRTbell template and can interfere with quantitation and polymerase binding.
- Has undergone a minimum of freeze-thaw cycles.
- Has not been exposed to high T (>65°C for 1 h can cause a detectable decrease in sequence quality).
- Has not been exposed to pH extremes (<6 or >9).
- Does not contain insoluble material, and is not colored or cloudy.
- Does not contain RNA.
- Has not been exposed to intercalating fluorescent dyes or UV radiation.
- Does not contain chelating agents (e.g., EDTA), divalent metal cations (like Mg 2+), denaturants (like guanidinium salts, phenol), or detergents (like SDS, Triton-X100).
- Does not contain carryover contamination from the starting organism/tissue (like heme, humic acid, polyphenols).