Preparation of sequencing libraries from DNA or cDNA for sequencing on an Illumina platform
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Documentation

The full protocol can be found here

What you’ll need

  • Tagment DNA buffer (TD) (Stored at -20C)
  • Amplicon Tagmentation Mix (ATM) (-20C)
  • Neutralization buffer (NT) (Room Temperature)
  • Nextera PCR Mix (NPM) (-20C)
  • Resuspension Buffer (RSB) (-20C)
  • TruSeq Index Plate Fixture (Room Temperature)
  • 80% Ethanol, made fresh the same day
  • Magnetic stand
  • AMPureXP beads (Beckman-Coulter)

A few important comments before you start

  • We use the Low Sample ‘LS’ protocol as we are typically working with fewer than 48 samples at a time
  • The recommended starting amount of total RNA is 100 ng - 1 ug, but we usually try to stay away from the extreme ends of this spectrum.
  • Split this protocol into two days, stopping on the first day after you have double stranded cDNA. On second day, do the A-tailing, adapter ligation, PCR, cleanup, and quality control steps.
  • For 12 samples, you will need approximately 6-7 hours on the first day, and 6-7 hours the second day.
  • Keep all reagents on ice unless otherwise stated.
  • We typically remove the reagents from storage during an incubation period in the previous step.
  • Have the beads at room temperature at least 30 minutes prior to use.
  • Aliquot the reagents that arrive in large quantities (such as the resuspension buffer and the bead washing buffer) into 2 mL Eppendorf tubes.
  • We do not perform any of the in-line controls.
  • We do not use the plate barcode stickers that come with the kit.
  • Use only filter pipette tips and clean your area so it is free of RNases.
  • Ensure that the AMPureXP beads are mixed well immediately prior to use.
  • There are many mixing steps in this protocol - I frequently use a multi-channel pipette to help speed up this process, particularly if many libraries are being prepared.
  • We use a Tapestation or a BioAnalyzer to assay the input RNA quality. Ribosomal integrity numbers of 7 and higher are preferred.
  • We quantify the input RNA using a Qubit fluorometer.

Step 1: Quantify DNA

Quantify DNA with a Qubit and then dilute each sample to 0.2 ng/uL using ultra-pure, molecular-grade water.

Step 2: Tagment DNA:

  • Add the following items in the order listed to each well of a new PCR plate. Pipette to mix:

10 uL Tagment DNA Buffer (TD) 5 uL Normalized gDNA

  • Add 5 μl ATM to each well. Pipette to mix. Seal the plate. The total volume should be 20 uL per well.

  • Centrifuge the plate at 280 × g at 20°C for 1 minute.

  • Place on the preprogrammed thermal cycler and run the tagmentation program.

  • Add 5 μl NT to each well. Pipette to mix.

  • Centrifuge at 280 × g at 20°C for 1 minute.

  • Incubate at room temperature for 5 minutes.

Step 3: PCR

  • Set up the indexes you will be using and arrange them in the TruSeq Index Plate Fixture (see manual for images).

  • Using a multichannel pipette, add 5 μl of each Index 1 (i7) adapter down each column.

  • Using a multichannel pipette, add 5 μl of each Index 2 (i5) adapter across each row.
  • Add 15 μl NPM to each well containing index adapters. Pipette to mix. The total volume is now 50 uL per well.

  • Centrifuge at 280 × g at 20°C for 1 minute.

  • Place on the preprogrammed thermal cycler and run the “NexteraPCR” program (bold denotes steps to be run for 12 cycles).

Temp (C) Time (min:sec)
72 3:00
95 0:30
95 0:10
55 0:30
72 0:30
72 5:00
10 hold

Step 4: Cleanup

  • Vortex AMPure XP beads before each use. Vortex AMPure XP beads frequently to make sure that beads are evenly distributed.

  • Add 30 μl AMPure XP beads to each well.

  • Shake at 1800 rpm for 2 minutes.

  • Incubate at room temperature for 5 minutes.

  • Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

  • Remove and discard all supernatant from each well.

  • Wash 2 times as follows:
  • Add 190 μl fresh 80% EtOH to each well.
  • Incubate on the magnetic stand for 30 seconds.
  • Remove and discard all supernatant from each well.

  • Using a 20 μl pipette, remove residual 80% EtOH from each well.

  • Air-dry on the magnetic stand for 15 minutes.

  • Remove from the magnetic stand.

  • Add 52.5 μl RSB to each well.

  • Shake at 1800 rpm for 2 minutes.

  • Incubate at room temperature for 2 minutes.

  • Place on a magnetic stand and wait until the liquid is clear (~2 minutes).

  • Transfer 50 μl supernatant to a new plate.

Step 5: Quality Check

  • Use the HS DNA5000 or HS DNA1000 tape and appropriate reagent buffer. A successful library preparation will have a broad peak with an average size between 400-1000 bp.

  • Quantify the sample with the HS DNA Qubit kit.