Quantification

All RNA and DNA quality control for library construction is done on an Agilent TapeStation 4150.

  • High Sensitivity RNA ScreenTape Assay
  • RNA ScreenTape Assay
  • High Sensitivity DNA 1000 ScreenTape Assay
  • DNA 1000 ScreenTape Assay

Libraries are quantified by qPCR on a Roche Lightcycler 96 using the KAPA Library Quantification kit to ensure equimolar pooling and proper loading concentrations prior to sequencing.

Library Preps

The latest library prep kits from Illumina and 10X Genomics are used.

  • Illumina Library Prep
    • Stranded Total RNA w/ Ribo-Zero Plus (rRNA depletion, 10-1000 ng input)
    • Stranded mRNA (poly-A capture, 25-1000 ng input)
    • DNA Prep (1-500 ng input)
    • PCR-Free DNA Prep (25-300 ng input)
  • 10X Genomics Single Cell Library Prep
    • Chromium Next GEM Single Cell 3' and 5'
    • ATAC and Multiome ATAC + Gene Expression

Sequencing

Sequencing is done on the Illumina NextSeq 1000. The NextSeq 1000 generates an average of 400M clusters which translates to ~400M single reads or ~800M paired end reads per run. There are currently three reagent cartridges available that offer varying read lengths.

  • P2 Cartridge with 100 Cycles: up to 50 bp paired end or 100 bp single read and 10X libraries
  • P2 Cartridge with 200 Cycles: up to 100 bp paired end or 150 bp single read
  • P2 Cartridge with 300 Cycles: up to 150 bp paired end

More Information

QuantificationLibrary PrepSequencing

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