When preparing sequencing-ready libraries, it is important to create an equimolar pool so that each library contributes equally to the final sequencing run. Below is the recommended workflow using the CUTANATM CUT&RUN Library Prep Kit (EpiCypher 14-1001).
Quantify each library and assess library size
Calculate the concentration of each library using the 200 - 700 bp range on the Agilent TapeStation® or Bioanalyzer®. Note that in most cases you will need to manually set this window.
Record the average fragment size (in base pairs) as determined by the TapeStation/BioAnalyzer.
Calculate molar concentration
Convert concentration (ng/µL) into molarity (nM) using the following formula (where 660 g/mol/bp is the approximate molecular weight of double-stranded DNA):
nM = (concentration in ng/µL × 1,000,000) ÷ (660 × average fragment size in bp)
Normalize libraries
Dilute each library to a common molarity using nuclease-free water or a low-EDTA buffer (such as 0.1X TE).
Pool normalized libraries
Combine equal volumes (e.g., 5 µL) of each normalized library into a single tube. This creates the equimolar pool.
Validate pooled libraries
Run the pool on a TapeStation or Bioanalyzer to confirm pooled fragment distribution. If needed, perform clean up for small fragments on the entire pooled library at this time.
Quantify the pool using Qubit.
Dilute to the concentration required for your sequencing platform.
Key Notes
Always normalize based on molarity, not mass concentration, to ensure equal sequencing representation.
If libraries vary widely in concentration, do NOT dilute to the lowest concentration sample.