View source for BioMicroCenter:PacBio Library Preparation

{{BioMicroCenter}}
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Pacific Biosciences offers a broad variety of methodologies for preparing libraries for sequencing with a primary library prep kit called SMRTBell Prep Kit v3.0. Pricing includes quality control prior to prep. 
Large batches will be undertaken on a custom basis; please email biomicro@mit.edu for more information.  

== dsDNA: Prep Kit v3.0 ==
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  !Parameter 
  !Requirements
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  |INPUT || Clean double-stranded DNA <BR> 150 ng-5 ug <BR> unsheared 50-60 kbp <br> >10uL
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  |INCLUDED || Initial QC by UV-vis and FemtoPulse <BR> Library preparation <BR> Final QC by Qubit
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  |SUBMISSION || MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863&tab=services ilabs] <BR> External - [[BioMicroCenter:Forms|form]]
  |-
  |UNIT || Per sample
  |-
  |PRICING ||[[BioMicroCenter:Pricing#LONG_READ_LIBRARIES|LINK]] 
  |}
  |
===Standard Workflow===
The BioMicro Center uses the SMRTBell Prep Kit v3.0 for dsDNA library preparation. This kit is recommended by PacBio for all libraries to be sequenced on the Revio. Single strand overhangs are removed, and DNA Damage repair is performed.  End-Repair and A-tailing proceed before SMRTBell A/T Ligation, after which two rounds of SPRI cleans with PacBio approved beads clean the prepared library.  Prepared libraries can be quantified via Qubit prior to polymerase binding.  If a size selection is necessary, the size will be verified by Fragment Analyzer or by Femto Pulse for sizes larger than 6 kbp.
|[[IMAGE:PacBioBound.jpg|500px|center|PacBio Library with Enzyme bound to SMRTBell hairpins<br>NOT TO SCALE]]
|}

== Genomic ==
Typically, a good extraction of genomic DNA that has been stored appropriately should have fragment sizes larger than 100 kbp, even for bacteria.  If sizes are smaller, it is likely that the DNA has been sheared somehow.  If the average size is determined to be 50-60 kbp, the standard workflow is followed.  If the average size is larger than 50-60kbp, the center has Covaris gTubes available for shearing to give a length slightly higher than 40 kbp.  For larger average read lengths, other shearing methods can be explored on a case by case basis.  After shearing and a potential size selection, the standard workflow proceeds.
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== IsoSeq ==
The standard IsoSeq protocol uses polyA RNA as input.  cDNA template is generated with the NEBNext® Single Cell/Low Input cDNA Synthesis and Amplification Module along with Template Switch Oligo from the PacBio Iso-Seq Express Oligo kit.  Template is amplified with the NEBNext® High-Fidelity 2X PCR Master Mix and Forward PCR primer from the PacBio Iso-Seq Express Oligo kit.  Enriching for longer transcripts requires longer elongation times for amplification. The normal protocol is then followed.