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Starting Material: RNA

Submitting Samples for RNA-Seq Library Prep & Sequencing

  1.  Consult with us on your project before making any requests. 
  2.  Determine which bulk RNA library preparation fits your needs.
    1. See the tabs below for sample concentration and volume requirements. 
  3.  Download our form, fill in all highlighted fields, and submit your form through minilims.
  4.  Array samples by column in a semi or full-skirted 96-well plate. Place samples in a provided zip-top bag with the completed submission slip.
  5.  Log your sample drop-off on the paper log.
  6.  Place the bag into the “sequencing drop-off" submission freezer in the hall.

Note: See here if you only want sequencing services.

ngs_rna_library_service_request_form_fy24.xlsx534 KB

RNA Library Preparations

KAPA mRNA HyperPrep Library Preparation

Using a SciClone G3 NGSx workstation (Perkin Elmer) the Core will prepare sequencing libraires using the Kapa mRNA Hyper Prep Kit (Roche Applied Science). 

  • Polyadenylated mRNAs captured from 50-500 ng of total RNA
  • Fragmentation to 300-400 bp using heat and magnesium 
  • First strand synthesis using random priming followed by second-strand synthesis and A-tailing
  • dUTP incorporation to allow strand-specific sequencing of the library
 
 
To submit samples for Kapa mRNA HyperPrep Library Preparation, please provide the following:
  • 60 ul of RNA between 1-10 ng/ul
    • Core will perform fluorometric assessment of input sample to verify concentration
    • Core can normalize input sample as a service
    • Samples below 50 ng input are not guaranteed 
  • RIN values for all samples
    • Core can perform TapeStation analysis as a service
    • RIN values <7 are not guaranteed
Other requirements:Image of 96-well plate with arrow down the first column
  • Samples must be arrayed in columns of a 96-well plate 
  • Samples and all appropriate Minilims requests must be submitted by the 15th of the month (or the Friday before as applicable)
    • If the core is completing QC on input samples submission date is the 8th of the month
    • Turnaround time is 4 weeks from the 15th of the month
  • Coordinate drop-off with Core Staff between 8 am – 4 pm

1/4 Smart-Seq v4 - Low Input, Bulk RNA

Using a Mantis Low Volume Liquid Dispenser (Formulatrix) and a Biomek FXP Span-8 Liquid Handler (Beckman Coulter) the Core will take input RNA samples and prepare cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and libraries using the Nextera XT DNA Library Prep Kit (Illumina). 

  • Switching Mechanism at 5′ End of RNA Template (SMART) technology captures full-length transcripts from 10 pg- 2.5 ng total RNA
  • Non-stranded full-length cDNAs, contains the 5' end of the mRNA transcript
  • Alternatively spliced isoform detection & rare low abundance transcripts
  • Tagmentation based library preparation

(Reference: SMART-Seq v4 Ultra Low Input RNA Kit- SSv4 050721)

 
To submit samples for 1/4 SMART-Seq v4 Library Preparation, please provide the following:
  • 10 ul of RNA between 0.00125 -1.25 ng/ul; Core will perform our own fluorometric assessment
  • RIN values for all samples; Core can perform TapeStation analysis as a service
    • RIN values <7 are not guaranteed
Image of 96-well plate with arrow down the first columnOther requirements:
  • Samples must be arrayed in columns of a 96-well plate 
  • Samples and all appropriate Minilims requests must be submitted by the 1st of the month (or the Friday before as applicable)
    • If the core is completing QC on input samples submission date is the 25th of the previous month
    • Turnaround time is 4 weeks from the 1st of the month
  • Coordinate drop-off with Core Staff between 8 am – 4 pm