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
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
epMotion 5075 TMX automated pipetting system with gripper
Dispensing tool TS 50
Dispensing tool TM 50-8
Dispensing tool TM 300-8
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)
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.