BBI-AGTC Services

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

Prior to library construction, input RNA and DNA samples are quantified using the Qubit 3 Fluorometer.

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

Library Preps

The latest library prep kits from Illumina, NEB, 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)
• NEB Library Prep
  • NEBNext Ultra II DNA (w/ fragmentation, 0.1 - 500 ng input)
  • NEBNext Ultra II FS DNA (w/o fragmentation, 0.5 - 1000 ng input)
• 10X Genomics Single Cell Library Prep
  • Chromium Next GEM Single Cell 3' and 5' Gene Expression
  • ATAC and Multiome ATAC + Gene Expression

Sequencing

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

• Yields ~100-130M reads:
  • P1 cartridge with 300 cycles: up to 150 bp paired end reads
• Yields ~400-500M reads:
  • 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 reads

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