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Automation of ChIP-Seq Library Preparation for Next Generation Sequencing on the epMotion® 5075 TMX
 
Cheng Liu, Ph.D.1, Jesse Cassidy1, and Maryke Appel, Ph.D.2
Full Text (PDF)

Materials and Methods

Kits and Reagents

  • Fragmented DNA (sheared to ~200 bp). Libraries were prepared from various quantities of input DNA, ranging between 1 ng and 200 ng. 1, 5, 10, and 20 ng samples were ChIP DNA, whereas 50 and 200 ng samples were cDNA.

  • Illumina™ TruSeq® Y-adapters

  • KAPA High-Throughput Library Preparation Kit (Cat. No. KK8234)

  • Agencourt® AMPure® XP beads (Beckman Coulter Cat. No. A63880)

  • Elution buffer (1× TE)

  • Freshly prepared 80% ethanol

Consumables

  • 1.5 mL Eppendorf DNA LoBind tubes

  • 0.1 mL Eppendorf PCR tubes

  • Eppendorf PCR Film (self-adhesive)

  • Eppendorf twin.tec PCR plates, semi-skirted

  • Eppendorf epT.I.P.S. Motion Filter 1–50 µL

  • Eppendorf epT.I.P.S. Motion Filter 20–300 µL

  • epMotion Reservoir 30 mL

  • epMotion Reservoir 100 mL

Equipment

  • epMotion 5075 TMX automated pipetting system with gripper

  • Dispensing tool TS 50

  • Dispensing tool TM 50-8

  • Dispensing tool TM 300-8

  • Reservoir rack

  • TC module for 1.5/2.0 mL tubes

  • Thermorack for 24 × 1.5/2.0 mL Safe-Lock tubes

  • Thermoblock for 96-well PCR plates

  • Eppendorf Mastercycler Pro

  • Agilent Technologies 2100 Bioanalyzer

  • Agencourt® SPRIPlate® Super Magnet Plate (Beckman Coulter Cat. No. A32782)

Procedure

Figure 1 outlines the essential steps of the workflow. Additional details are provided below.

  • The epMotion was programmed according to the instructions provided in the KAPA High-Throughput Library Preparation Kit Technical Data Sheet. The epMotion worktable setup is shown in Figure 2.

  • The recommended second post-ligation cleanup was performed to completely remove un-ligated adapters (see section 6 of KAPA HTP Library Preparation Kit Technical Data Sheet). DNA was eluted into 30 µL of elution buffer after this step. About 25% (7 µL) was set aside for QC and/or troubleshooting and the remaining 23 µL were use for library amplification.

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